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Professor Michael Geeves
Professor of Physical Biochemistry
School of Biosciences
- M.A.Geeves@kent.ac.uk
- 01227 (82)7597
Professor Mike Geeves joined the School of Biosciences in April 1999. He studied biochemistry as an undergraduate at the University of Birmingham then went on to the University of Bristol to work on a PhD with David Trentham. It was here that he first came to work on the myosin motor which has been the focus of his work ever since. In those early days it was muscle myosin - the only known form of myosin. After completing his PhD he spent 2 years at the University of California, Santa Cruz studying enzymology at sub-zero temperatures with Anthony Fink. He then return to spend 14 years at the University of Bristol working alongside Freddie Gutfreund, first as an SERC Junior Fellow then as a Royal Society University Fellow. At the end of the fellowship he moved to become a Group Leader in the new Max Planck Institute of Molecular Physiology that was being established in Dortmund by Roger Goody. He left there to take up the current position as Professor of Physical Biochemistry.
Mike is a member of the Mechanobiology group also known as MaDCaP.
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Article
Brooker, H. et al. (2018). A novel live cell imaging system reveals a reversible hydrostatic pressure impact on cell cycle progression. Journal of Cell Science [Online]. Available at: http://dx.doi.org/10.1242/jcs.212167.
Life is dependent upon the ability of a cell to rapidly respond to changes in
environment. Small perturbations in local environments change the ability of
molecules to interact and hence communicate. Hydrostatic pressure provides
a rapid non-invasive, fully-reversible method for modulating affinities between
molecules both in vivo and in vitro. We have developed a simple fluorescence
imaging chamber that allows intracellular protein dynamics and molecular
events to be followed at pressures up to 200 bar in living cells. Using yeast we
investigate the impact of hydrostatic pressure upon cell growth and cell cycle
progression. While 100 bar has no affect upon viability, it induces a delay in
chromosome segregation, resulting in the accumulation of long-undividedbent
cells, consistent with disruption of the cytoskeletons. This delay is
independent of stress signalling and induces synchronisation of cell-cycle
progression. Equivalent affects were observed in Candida albicans, with
pressure inducing a reversible cell-cycle delay and hyphal growth. We present
a simple novel non-invasive fluorescence microscopy based approach to
transiently impact molecular dynamics to visualise, dissect and study signalling pathways and cellular processes in living cells.
Geeves, M. et al. (2018). Dilated cardiomyopathy myosin mutants have reduced force-generating capacity. Journal of Biological Chemistry [Online]. Available at: http://dx.doi.org/10.1074/jbc.RA118.001938.
Abstract | View in KAR | View Full Text
Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human β-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M.D complex in the steady-state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force holding capacity due to the reduced occupancy of the force-holding state.
Karatzaferi, C., Adamek, N. and Geeves, M. (2017). Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue. American Journal of Physiology - Cell Physiology [Online] 313:C644-C654. Available at: http://dx.doi.org/10.1152/ajpcell.00023.2017.
The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define 'fatigability' in vivo of fast vs slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin sub-fragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a pre-equilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs pH 6.2) and Pi (zero, 15 or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1 but for slow S1 KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.
Geeves, M. (2017). More Can Mean Less, or: Simplifying Sometimes Requires Ideas To Be More Complicated. Biophysical Journal [Online] 112:2467-2468. Available at: http://dx.doi.org/10.1016/j.bpj.2017.05.004.
Mijailovich, S. et al. (2017). Modeling the Actin.myosin ATPase cross-bridge cycle for skeletal and cardiac muscle myosin isoforms. Biophysical Journal [Online] 112:984-996. Available at: http://dx.doi.org/10.1016/j.bpj.2017.01.021.
Abstract | View in KAR | View Full Text
Modeling the complete actin.myosin ATPase cycle has always been limited by the lack of experimental data concerning key steps of the cycle, because these steps can only be defined at very low ionic strength. Here, using human β-cardiac myosin-S1, we combine published data from transient and steady-state kinetics to model a minimal eight-state ATPase cycle. The model illustrates the occupancy of each intermediate around the cycle and how the occupancy is altered by changes in actin concentration for [actin] = 1–20Km. The cycle can be used to predict the maximal velocity of contraction (by motility assay or sarcomeric shortening) at different actin concentrations (which is consistent with experimental velocity data) and predict the effect of a 5 pN load on a single motor. The same exercise was repeated for human α-cardiac myosin S1 and rabbit fast skeletal muscle S1. The data illustrates how the motor domain properties can alter the ATPase cycle and hence the occupancy of the key states in the cycle. These in turn alter the predicted mechanical response of the myosin independent of other factors present in a sarcomere, such as filament stiffness and regulatory proteins. We also explore the potential of this modeling approach for the study of mutations in human β-cardiac myosin using the hypertrophic myopathy mutation R453C. Our modeling, using the transient kinetic data, predicts mechanical properties of the motor that are compatible with the single-molecule study. The modeling approach may therefore be of wide use for predicting the properties of myosin mutations.
Johnson, C. et al. (2017). Temperature sensitive point mutations in fission yeast tropomyosin have long range effects on the stability and function of the actin- tropomyosin copolymer. Biochemical and Biophysical Research Communications [Online]. Available at: https://doi.org/10.1016/j.bbrc.2017.10.109.
Abstract | View in KAR | View Full Text
The actin cytoskeleton is modulated by regulatory actin-binding proteins which fine- tune the dynamic properties of the actin polymer to regulate function. One such actin-binding protein is tropomyosin (Tpm), a highly-conserved alpha-helical dimer which stabilises actin and regulates interactions with other proteins. Temperature sensitive mutants of Tpm are invaluable tools in the study of actin filament dependent processes, critical to the viability of a cell. Here we investigated the molecular basis of the temperature sensitivity of fission yeast Tpm mutants which fail to undergo cytokinesis at the restrictive temperatures. Comparison of Contractile Actomyosin Ring (CAR) constriction as well as cell shape and size revealed the cdc8.110 or cdc8.27 mutant alleles displayed significant differences in their temperature sensitivity and impact upon actin dependent functions during the cell cycle. In vitro analysis revealed the mutant proteins displayed a different reduction in thermostability, and unexpectedly yield two discrete unfolding domains when acetylated on their amino-termini. Our findings demonstrate how subtle changes in structure (point mutations or acetylation) alter the stability not simply of discrete regions of this conserved cytoskeletal protein but of the whole molecule. This differentially impacts the stability and cellular organisation of this essential cytoskeletal protein.
Walklate, J., Ujfalusi, Z. and Geeves, M. (2016). Myosin isoforms and the mechanochemical cross-bridge cycle. Journal of Experimental Biology [Online] 219:168-174. Available at: http://doi.org/10.1242/jeb.124594.
At the latest count the myosin family includes 35 distinct groups, all of which have the conserved myosin motor domain attached to a neck or lever arm, followed by a highly variable tail or cargo binding region. The motor domain has an ATPase activity that is activated by the presence of actin. One feature of the myosin ATPase cycle is that it involves an association/dissociation with actin for each ATP hydrolysed. The cycle has been described in detail for a large number of myosins from different classes. In each case the cycle is similar, but the balance between the different molecular events in the cycle has been altered to produce a range of very different mechanical activities. Myosin may spend most of the ATPase cycle attached to actin (high duty ratio), as in the processive myosin (e.g. myosin V) or the strain-sensing myosins (e.g. myosin 1c). In contrast, most muscle myosins spend 80% of their ATPase cycle detached from actin. Within the myosin IIs found in human muscle, there are 11 different sarcomeric myosin isoforms, two smooth muscle isoforms as well as three non-muscle isoforms. We have been exploring how the different myosin isoforms have adapted the cross-bridge cycle to generate different types of mechanical activity and how this goes wrong in inherited myopathies. The ideas are outlined here.
Geeves, M. (2016). The ATPase mechanism of myosin and actomyosin. Biopolymers [Online] 105:483-491. Available at: http://doi.org/10.1002/bip.22853.
Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. This article is protected by copyright. All rights reserved.
Mijailovich, S. et al. (2016). Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice. The Journal of General Physiology [Online] 148:459-488. Available at: http://doi.org/10.1085/jgp.201611608.
Walklate, J. et al. (2016). The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects. Journal of Biological Chemistry [Online] 291:10318-10331. Available at: http://doi.org/10.1074/jbc.M115.707489.
The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5–9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2–35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.
Brooker, H., Geeves, M. and Mulvihill, D. (2016). Analysis of biophysical and functional consequences of Tropomyosin - fluorescent protein fusions. FEBS letters [Online]:3111-3121. Available at: http://onlinelibrary.wiley.com/doi/10.1002/1873-3468.12346/full.
Abstract | View in KAR | View Full Text
The dynamic nature of actin polymers is modulated to facilitate a diverse
range of cellular processes. These dynamic properties are modulated by
different isoforms of Tropomyosin, which are recruited to distinct subpopulations
of actin polymers to differentially modulate their functional
properties. This makes them an attractive target for labelling discrete actin
populations. We have assessed the effect of different fluorescent labelling
strategies for this protein. Although tropomyosin fluorescent fusions
decorate actin in vivo, they are either non-functional or perturb regulation
of actin nucleation and cell cycle timings. Thus conclusions and
physiological relevance should be carefully evaluated when using
tropomyosin fusions.
Nag, S. et al. (2015). Contractility parameters of human -cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function. Science Advances [Online] 1:e1500511-e1500511. Available at: http://doi.org/10.1126/sciadv.1500511.
Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation.
Lehman, W. et al. (2015). Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain. Archives of Biochemistry and Biophysics [Online] 571:10-15. Available at: http://doi.org/10.1016/j.abb.2015.02.026.
The ends of coiled-coil tropomyosin molecules are joined together by nine to ten residue-long head-to-tail "overlapping domains". These short four-chained interconnections ensure formation of continuous tropomyosin cables that wrap around actin filaments. Molecular Dynamics simulations indicate that the curvature and bending flexibility at the overlap is 10–20% greater than over the rest of the molecule, which might affect head-to-tail filament assembly on F-actin. Since the penultimate residue of striated muscle tropomyosin, Ser283, is a natural target of phosphorylating enzymes, we have assessed here if phosphorylation adjusts the mechanical properties of the tropomyosin overlap domain. MD simulations show that phosphorylation straightens the overlap to match the curvature of the remainder of tropomyosin while stiffening it to equal or exceed the rigidity of canonical coiled-coil regions. Corresponding EM data on phosphomimetic tropomyosin S283D corroborate these findings. The phosphorylation-induced change in mechanical properties of tropomyosin likely results from electrostatic interactions between C-terminal phosphoSer283 and N-terminal Lys12 in the four-chain overlap bundle, while promoting stronger interactions among surrounding residues and thus facilitating tropomyosin cable assembly. The stiffening effect of D283-tropomyosin noted correlates with previously observed enhanced actin–tropomyosin activation of myosin S1-ATPase, suggesting a role for the tropomyosin phosphorylation in potentiating muscle contraction.
Geeves, M., Hitchcock-DeGregori, S. and Gunning, P. (2015). A systematic nomenclature for mammalian tropomyosin isoforms. Journal of Muscle Research and Cell Motility [Online] 36:147-153. Available at: http://doi.org/10.1007/s10974-014-9389-6.
Tropomyosin, a ubiquitous protein in animals and fungi, is associated with the actin cytoskeleton and is involved with stabilising actin filaments and regulating the interaction of the filament with other actin binding proteins. The protein is best known for its role in regulating the interaction between actin and myosin in muscle contraction but in recent years its role as a major player in the organisation and dynamics of the cytoskeleton has been increasingly recognised. In mammals Tpm is expressed from four distinct genes and alternate splicing of each gene can produce a total of up to 40 different mRNA variants most of which are expressed as proteins. We are expecting a renaissance in the study of tropomyosins as the roles of these different isoforms are beginning to be deciphered. However, it is our belief that such a renaissance is being limited by confusion over the naming systems for the tropomyosin isoforms. These result in even experienced workers struggling to reconcile work done in different laboratories and at different times. We propose here a systematic nomenclature for tropomyosin based on the best current practice. We recommend the adoption of these names and a cross-reference to the table of alternate names and accession numbers for protein sequences is included here. The National Center for Biotechnology Information (NCBI) website has been amended to include the nomenclature for the human, mouse and rat genes.
Desai, R., Geeves, M. and Kad, N. (2015). Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments. Journal of Biological Chemistry [Online]:jbc.M114.609743-jbc.M114.609743. Available at: http://dx.doi.org/10.1074/jbc.M114.609743.
Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin's access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here we directly image single molecules of myosin binding to and activating thin filaments. Using this approach the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 further myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off on a binary fashion.
Bloemink, M. et al. (2015). The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II. Journal of Biological Chemistry [Online] 291:1763-1773. Available at: http://doi.org/10.1074/jbc.M115.688002.
The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.
Walklate, J. and Geeves, M. (2015). Temperature manifold for a stopped-flow machine to allow measurements from −10 to +40°C. Analytical Biochemistry [Online] 476:11-16. Available at: http://doi.org/10.1016/j.ab.2015.01.020.
Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 °C or >30 °C) can damage the machine or the enzyme. We have devised a simple manifold that can be attached to most commercial stopped-flow systems that is independently heated or cooled separate from the main stopped-flow system. Careful calibration of the flow circuit allows the sample to be heated or cooled to the measurement temperature (−8 to +40 °C) 1 to 2 s before mixing in the reaction chamber. This approach allows measurements at temperatures where the stopped flow or the protein is normally unstable. To validate the manifold, we investigated the well-defined ATP-induced dissociation of rabbit muscle myosin subfragment 1 (S1) from its complex with pyrene-labeled actin. This process has both temperature-dependent and -independent components. Use of ethylene glycol allowed us to measure the reaction below 0 °C and up to 42 °C, and as expected the second-order rate constant (K1k+2) and the maximum rate of dissociation (k+2) both increased with temperature, whereas 1/K1 is unaffected by the change in temperature.
Bloemink, M. et al. (2014). The Hypertrophic Cardiomyopathy Myosin Mutation R453C Alters ATP Binding and Hydrolysis of Human Cardiac beta-Myosin. Journal of Biological Chemistry [Online] 289:5158-5167. Available at: http://dx.doi.org/10.1074/jbc.M113.511204.
The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long α-helix, helix O, and to Switch-2 via the fifth strand of the central β-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecular activities. We report here the first detailed biochemical kinetic analysis of the motor domain of the human β-cardiac myosin carrying the R453C mutation. A recent report of the same mutation (Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., and Spudich, J. A. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 12607–12612) found reduced ATPase and in vitro motility but increased force production using an optical trap. Surprisingly, our results show that the mutation alters few biochemical kinetic parameters significantly. The exceptions are the rate constants for ATP binding to the motor domain (reduced by 35%) and the ATP hydrolysis step/recovery stroke (slowed 3-fold), which could be the rate-limiting step for the ATPase cycle. Effects of the mutation on the recovery stroke are consistent with a perturbation of Switch-2 closure, which is required for the recovery stroke and the subsequent ATP hydrolysis.
Geeves, M. and Lehrer, S. (2014). Cross-Talk, Cross-Bridges, and Calcium Activation of Cardiac Contraction. Biophysical Journal [Online] 107:543-545. Available at: http://dx.doi.org/10.1016/j.bpj.2014.06.019.
Lehrer, S. and Geeves, M. (2014). The myosin-activated thin filament regulatory state, M − -open: a link to hypertrophic cardiomyopathy (HCM). Journal of Muscle Research and Cell Motility [Online] 35:153-160. Available at: http://doi.org/10.1007/s10974-014-9383-z.
Lawrence, A. et al. (2014). FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR). Bioscience Reports [Online] 34:345-355. Available at: http://dx.doi.org/10.1042/BSR20140060.
Abstract | View in KAR | View Full Text
Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I).
Bloemink, M. et al. (2013). The Superfast Human Extraocular Myosin Is Kinetically Distinct from the Fast Skeletal IIa, IIb, and IId Isoforms. Journal of Biological Chemistry [Online] 288:27469-27479. Available at: http://dx.doi.org/10.1074/jbc.M113.488130.
Janco, M. et al. (2013). Polymorphism in tropomyosin structure and function. Journal of Muscle Research and Cell Motility [Online] 34:177-187. Available at: http://dx.doi.org/10.1007/s10974-013-9353-x.
Tropomyosins (Tm) in humans are expressed from four distinct genes and by alternate splicing >40 different Tm polypeptide chains can be made. The functional Tm unit is a dimer of two parallel polypeptide chains and these can be assembled from identical (homodimer) or different (heterodimer) polypeptide chains provided both chains are of the same length. Since most cells express multiple isoforms of Tm, the number of different homo and heterodimers that can be assembled becomes very large. We review the mechanism of dimer assembly and how preferential assembly of some heterodimers is driven by thermodynamic stability. We examine how in vitro studies can reveal functional differences between Tm homo and heterodimers (stability, actin affinity, flexibility) and the implication for how there could be selection of Tm isomers in the assembly on to an actin filament. The role of Tm heterodimers becomes more complex when mutations in Tm are considered, such as those associated with cardiomyopathies, since mutations can appear in only one of the chains.
Li, X. et al. (2012). The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy. Biochemical and Biophysical Research Communications [Online] 424:493-496. Available at: http://dx.doi.org/10.1016/j.bbrc.2012.06.141.
Canepari, M. et al. (2012). Actomyosin kinetics of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach. Experimental Physiology [Online] 97:873-881. Available at: http://dx.doi.org/10.1113/expphysiol.2012.064576.
An in vitro motility assay approach was used to investigate the mechanisms of the functional differences between myosin isoforms, by studying the effect of MgATP and MgADP on actin sliding velocity (Vf) of pure slow and fast rat skeletal myosin at different temperatures. The value of Vf depended on [MgATP] according to Michaelis–Menten kinetics, with an apparent constant (Km) of 54.2, 64.4 and 200 μM for the fast isoform and 18.6, 36.5 and 45.5 μM for the slow isoform at 20, 25 and 35°C, respectively. The presence of 2 mM MgADP decreased Vf and yielded an inhibition constant (Ki) of 377, 463 and 533 μM for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 μM for the slow isoform at 25 and 35°C, respectively. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover, the higher sensitivity of the fast myosin isoform to a drop in [MgATP] is consistent with the higher fatigability of fast fibres than slow fibres. From the Michaelis–Menten relation in the absence of MgADP, we calculated the rate of actomyosin dissociation by MgATP (k+ATP) and the rate of MgADP release (k-ADP). We found values of k+ATP of 4.8 × 106, 6.5 × 106 and 6.6 × 106 M−1 s−1 for the fast isoform and 3.3 × 106, 2.9 × 106 and 6.7 × 106 M−1 s−1 for the slow isoform and values of k-ADP of 263, 420 and 1320 s−1 for the fast isoform and 62, 107 and 306 s−1 for the slow isoform at 20, 25 and 35°C, respectively. The results suggest that k-ADP could be the major determinant of functional differences between the fast and slow myosin isoforms at physiological temperatures.
Geeves, M. (2012). 4.13 Thin Filament Regulation. Comprehensive Biophysics [Online] 4:251-267. Available at: http://dx.doi.org/10.1016/B978-0-12-374920-8.00416-1.
The review summarizes the current state of knowledge of the calcium regulation of striated muscle contraction via the thin filament proteins, tropomyosin and troponin. The description focuses on in vitro studies of the thin filament and covers structural, biochemical and dynamic aspects of the thin filament's response to calcium binding. A reductionist approach has allowed many of the transitions to be defined at the level of a single structural unit. Here an emphasis is placed on the co-operative nature of the structural and biochemical transitions of the thin filament and the allosteric relationship between calcium and myosin binding to the thin filament.
Mijailovich, S. et al. (2012). The Hill Model for Binding Myosin S1 to Regulated Actin Is not Equivalent to the McKillop–Geeves Model. Journal of Molecular Biology [Online] 417:112-128. Available at: http://dx.doi.org/10.1016/j.jmb.2012.01.011.
The Hill two-state cooperativity model and the McKillop–Geeves (McK–G) three-state model predict very similar binding traces of myosin subfragment 1 (S1) binding to regulated actin filaments in the presence and absence of calcium, and both fit the experimental data reasonably well [Chen et al., Biophys. J., 80, 2338–2349]. Here, we compared the Hill model and the McK–G model for binding myosin S1 to regulated actin against three sets of experimental data: the titration of regulated actin with S1 and the kinetics of S1 binding of regulated actin with either excess S1 to actin or excess actin to S1. Each data set was collected for a wide range of specified calcium concentrations. Both models were able to generate reasonable fits to the time course data and to titration data. The McK–G model can fit all three data sets with the same calcium-concentration-sensitive parameters. Only KB and KT show significant calcium dependence, and the parameters have a classic pCa curve. A unique set of the Hill model parameters was extremely difficult to estimate from the best fits of multiple sets of data. In summary, the McK–G cooperativity model more uniquely resolves parameters estimated from kinetic and titration data than the Hill model, predicts a sigmoidal dependence of key parameters with calcium concentration, and is simpler and more suitable for practical use.
Deery, E. et al. (2012). An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis. Nature Chemical Biology [Online] 8:933-940. Available at: http://dx.doi.org/10.1038/nchembio.1086.
The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B 12) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.
Mijailovich, S. et al. (2012). Cooperative regulation of myosin-S1 binding to actin filaments by a continuous flexible Tm–Tn chain. European Biophysics Journal [Online] 41:1015-1032. Available at: http://dx.doi.org/10.1007/s00249-012-0859-8.
The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC's degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k −I. The resulting equilibrium constant KB≡1/KI varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa <5.5) with a Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations.
Janco, M. et al. (2012). α-Tropomyosin with a D175N or E180G Mutation in Only One Chain Differs from Tropomyosin with Mutations in Both Chains. Biochemistry [Online] 51:9880-9890. Available at: http://dx.doi.org/10.1021/bi301323n.
α-Tropomyosin (Tm) carrying hypertrophic cardiomyopathy mutation D175N or E180G was expressed in Escherichia coli. We have assembled dimers of two polypeptide chains in vitro that carry one (αα*) or two (α*α*) copies of the mutation. We found that the presence of the mutation has little effect on dimer assembly, thereby predicting that individuals heterozygous for the Tm mutations are likely to express both αα* and α*α* Tm. Depending on the expression level, the heterodimer may be the predominant form in individuals carrying the mutation. Thus, it is important to define differences in the properties of Tm molecules carrying one or two copies of the mutation. We examined the Tm homo- and heterodimer properties: actin affinity, thermal stability, calcium regulation of myosin subfragment 1 binding, and calcium regulation of myofibril force. We report that the properties of the heterodimer may be similar to those of the wild-type homodimer (actin affinity, thermal stability, D175N αα*), similar to those of the mutant homodimer (calcium sensitivity, D175N αα*), intermediate between the two (actin affinity, E180G αα*), or different from both (thermal stability, E180G αα*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.
Kalyva, A., Schmidtmann, A. and Geeves, M. (2012). In Vitro Formation and Characterization of the Skeletal Muscle α·β Tropomyosin Heterodimers. Biochemistry [Online] 51:6388-6399. Available at: http://dx.doi.org/10.1021/bi300340r.
Tropomyosin (Tm) is a dimer made of two alpha helical chains associated into a parallel coiled-coil. In mammalian skeletal and cardiac muscle, the Tm is expressed from two separate genes to give the α- and β-Tm isoforms. These associate in vivo to form homo- (α2) and heterodimers (α·β) with little β2 normally observed. The proportion of α2 vs α·β varies across species and across muscle types from almost 100% α2- to 50% α·β-Tm. The ratio can also vary during development and in disease. The functional significance of the presence of these two isoforms has not been defined because it is difficult to isolate or purify the α·β dimer for functional studies. Here we report an effective method for purifying bacterially expressed Tm as α·β dimers using a cleavable N-terminal tag on one of the two chains. The same method can be used to isolate Tm dimers in which one chain carries a mutation. We go on to show that the α·β dimers differ in key properties (actin affinity, thermal stability) from either the α2- or β2-Tm. However, the ability to regulate myosin binding when combined with cardiac troponin appears unaffected.
Deacon, J. et al. (2012). Identification of functional differences between recombinant human α and β cardiac myosin motors. Cellular and Molecular Life Sciences [Online] 69:2261-2277. Available at: http://dx.doi.org/10.1007/s00018-012-0927-3.
Abstract | View in KAR | View Full Text
The myosin isoform composition of the heart is
dynamic in health and disease and has been shown to affect
contractile velocity and force generation. While different
mammalian species express different proportions of a and
b myosin heavy chain, healthy human heart ventricles
express these isoforms in a ratio of about 1:9 (a:b) while
failing human ventricles express no detectable a-myosin.
We report here fast-kinetic analysis of recombinant human
a and b myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin
motor and the first of a-myosin from any species. Our
findings reveal substantial isoform differences in individual
kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, a-subfragment 1
(S1) is far more similar to adult fast skeletal muscle myosin
isoforms than to the slow b isoform despite 91% sequence
identity between the motor domains of a- and b-myosin.
Among the features that differentiate a- from b-S1: the
ATP hydrolysis step of a-S1 is *ten-fold faster than b-S1,
a-S1 exhibits *five-fold weaker actin affinity than b-S1,
and actin a-S1 exhibits rapid ADP release, which is > tenfold faster than ADP release for b-S1. Overall, the cycle
times are ten-fold faster for a-S1 but the portion of time
each myosin spends tightly bound to actin (the duty ratio)
is similar. Sequence analysis points to regions that might
underlie the basis for this finding.
Deacon, J. et al. (2012). Erratum to: Identification of functional differences between recombinant human α and β cardiac myosin motors. Cellular and Molecular Life Sciences [Online] 69:4239-4255. Available at: http://dx.doi.org/10.1007/s00018-012-1111-5.
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.
Geeves, M. and Ranatunga, K. (2012). Tuning the Calcium Sensitivity of Cardiac Muscle. Biophysical Journal [Online] 103:849-850. Available at: http://dx.doi.org/10.1016/j.bpj.2012.07.039.
Rayes, R. et al. (2011). Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe. PLoS ONE [Online] 6:e21277. Available at: http://dx.doi.org/10.1371/journal.pone.0021277.
The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.
Geeves, M. et al. (2011). Cooperative [Ca²+]-dependent regulation of the rate of myosin binding to actin: solution data and the tropomyosin chain model. Biophysical Journal [Online] 100:2679-2687. Available at: http://dx.doi.org/10.1016/j.bpj.2011.04.020.
The regulation of muscle contraction by calcium involves interactions among actin filaments, myosin-S1, tropomyosin (Tm), and troponin (Tn). We have extended our previous model in which the TmTn regulatory units are treated as a continuous flexible chain, and applied it to transient kinetic data. We have measured the time course of myosin-S1 binding to actin-Tm-Tn filaments in solution at various calcium levels with [actin]/[myosin] ratios of 10 and 0.1, which exhibit modest slowing as [Ca(2+)] is reduced and a lag phase at low calcium. These observations can be explained if myosin binds to actin in two steps, where the first step is rate-limiting and blocked by TmTnI at low calcium, and the second step is fast, reversible, and controlled by the neighboring configuration of coupled tropomyosin-troponin units. The model can describe the calcium dependence of the observed myosin binding reactions and predicts cooperative calcium binding to TnC with competition between actin and Ca-TnC for the binding of TnI. Implications for theories of thin-filament regulation in muscle are discussed.
Adamek, N., Geeves, M. and Coluccio, L. (2011). Myo1c mutations associated with hearing loss cause defects in the interaction with nucleotide and actin. Cellular and Molecular Life Sciences [Online] 68:139-150. Available at: http://dx.doi.org/10.1007/s00018-010-0448-x.
Three heterozygous missense mutations in the motor domain of myosin 1c (Myo1c), which mediates adaptation in the inner ear, are associated with bilateral sensorineural hearing loss in humans. With transient kinetic analyses, steady-state ATPase and motility assays, and homology modeling, we studied the interaction of these mutants with nucleotide and actin using a truncated construct, Myo1c(1IQ-SAH), which includes an artificial lever arm. Results indicate that mutation R156W, near switch 1, affects the nucleotide-binding pocket and the calcium binding by disrupting switch 1 movement. Mutation V252A, in the K helix of the upper 50 kDa domain, showed reduced actin affinity consistent with disruption of communication between the actin- and nucleotide-binding sites. T380M, in a Myo1c-specific insert in the HO linker, displayed aberrant changes in most kinetic parameters and uncoupling of the ATPase from motility. These data allow for an interpretation of how these mutations might affect adaptation.
Bloemink, M. and Geeves, M. (2011). Shaking the myosin family tree: Biochemical kinetics defines four types of myosin motor. Seminars in Cell & Developmental Biology [Online] 22:961-967. Available at: http://dx.doi.org/10.1016/j.semcdb.2011.09.015.
Although all myosin motors follow the same basic cross-bridge cycle, they display a large variety in the rates of transition between different states in the cycle, allowing each myosin to be finely tuned for a specific task. Traditionally, myosins have been classified by sequence analysis into a large number of sub-families (∼35). Here we use a different method to classify the myosin family members which is based on biochemical and mechanical properties. The key properties that define the type of mechanical activity of the motor are duty ratio (defined as the fraction of the time myosin remains attached to actin during each cycle), thermodynamic coupling of actin and nucleotide binding to myosin and the degree of strain-sensitivity of the ADP release step. Based on these properties we propose to classify myosins into four different groups: (I) fast movers, (II) slow/efficient force holders, (III) strain sensors and (IV) gates.
Preller, M. et al. (2011). Structural Basis for the Allosteric Interference of Myosin Function by Reactive Thiol Region Mutations G680A and G680V. Journal of Biological Chemistry [Online] 286:35051-35060. Available at: http://dx.doi.org/10.1074/jbc.M111.265298.
The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures.
Korte, F. et al. (2011). Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP, contractility, and relaxation. Journal of Molecular and Cellular Cardiology [Online] 51:894-901. Available at: http://dx.doi.org/10.1016/j.yjmcc.2011.08.026.
We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca2+-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca2+ fluorescence were monitored by video microscopy. At 0.5 Hz stimulation, the extent of shortening was increased ~ 40% and maximal rate of shortening was increased ~ 80% in cardiomyocytes overexpressing Rrm1 + Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~ 150% with Rrm1 + Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1 + Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca2+], indicating increased contractility is primarily due to increased myofilament activity without altering Ca2+ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1 + Rrm2 overexpression as stimulation frequency was increased (1 Hz and 2 Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~ 1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca2+ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin–myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca2+. This article is part of a Special issue entitled "Possible Editorial".
Bloemink, M. et al. (2011). Two Drosophila myosin transducer mutants with distinct cardiomyopathies have divergent ADP and actin affinities. Journal of Biological Chemistry [Online] 286:28435-28443. Available at: http://dx.doi.org/10.1074/jbc.M111.258228.
Two Drosophila myosin II point mutations (D45 and Mhc(5)) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc(5)) could stabilize (D45) or destabilize (Mhc(5)) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1(A261T) and S1(G200D)) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1(A261T) is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin · S1), and reduced ATP-induced dissociation of actin · S1), whereas S1(G200D) shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin · S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1(A261T) (dilated) and S1(G200D) (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1(A261T) weakens the affinity of S1 · ADP for actin, whereas S1(G200D) increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up(2) mutation in Drosophila.
Mijailovich, S. et al. (2010). Resolution and uniqueness of estimated parameters of a model of thin filament regulation in solution. Computational Biology and Chemistry [Online] 34:19-33. Available at: http://dx.doi.org/10.1016/j.compbiolchem.2009.11.002.
The estimation of chemical kinetic rate constants for any non-trivial model is complex due to the nonlinear effects of second order chemical reactions. We developed an algorithm to accomplish this goal based on the Damped Least Squares (DLS) inversion method and then tested the effectiveness of this method on the McKillop–Geeves (MG) model of thin filament regulation. The kinetics of MG model is defined by a set of nonlinear ordinary differential equations (ODEs) that predict the evolution of troponin–tropomyosin–actin and actin–myosin states. The values of the rate constants are estimated by integrating these ODEs numerically and fitting them to a series of stopped-flow pyrene fluorescence transients of myosin-S1 fragment binding to regulated actin in solution. The accuracy and robustness of the estimated rate constants are evaluated for DLS and two other methods, namely quasi-Newton (QN) and simulated annealing (SA). The comparison of these methods revealed that SA provides the best estimates of the model parameters because of its global optimization scheme. However it converges slowly and does quantify the uniqueness of the estimated parameters. On the other hand the QN method converges rapidly but only if the initial guess of the parameters is close to the optimum values, otherwise it diverges. Overall, the DLS method proves to be the most convenient method. It converges fast and was able to provide excellent estimates of kinetic parameters. Furthermore, DLS provides the model resolution matrix, which quantifies the interdependence of model parameters thereby evaluating the uniqueness of their estimated values. This property is essential for estimating of the dependence of the model parameters on experimental conditions (e.g. Ca2+ concentration) when it is assessed from noisy experimental data such as pyrene fluorescence from stopped-flow transients. The advantages of the DLS method observed in this study should be further examined in other physicochemical systems to firmly establish the observed effectiveness of DSL vs. the other parameter estimation methods.
Johnson, M. et al. (2010). Targeted amino-terminal acetylation of recombinant proteins in E. coli. PLoS ONE [Online] 5:e15801. Available at: http://dx.plos.org/10.1371/journal.pone.0015801.
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One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co- expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.
Adamek, N. et al. (2010). Modification of Loop 1 Affects the Nucleotide Binding Properties of Myo1c, the Adaptation Motor in the Inner Ear. Biochemistry [Online] 49:958-971. Available at: http://dx.doi.org/10.1021/bi901803j.
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Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c1IQ, as well as chimeras of Myo1c1IQ with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties, or Ca2+ sensitivity of Myo1c1IQ. Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c1IQ-tonic) or replacement with a single glycine (Myo1c1IQ-G) accelerated the release of ADP from A.M 2−3-fold in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c1IQ-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single α-helix, or SAH, domain showed that Myo1cSAH-tonic translocated actin in vitro twice as fast as Myo1cSAH-WT and 3-fold faster than Myo1cSAH-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras.
Jenkins, D. et al. (2009). Rapid folding of the prion protein captured by pressure-jump. European Biophysics Journal [Online] 38:625-635. Available at: http://dx.doi.org/10.1007/s00249-009-0420-6.
The conversion of the cellular form of the prion protein (PrP(C)) to an altered disease state, generally denoted as scrapie isoform (PrP(Sc)), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions. Equilibrium folding was studied by circular dichroism and fluorescence. Pressure-jump experiments were successfully applied to reveal rapid submillisecond folding events of PrP at temperatures not accessed before.
Albet-Torres, N. et al. (2009). Drug Effect Unveils Inter-head Cooperativity and Strain-dependent ADP Release in Fast Skeletal Actomyosin. Journal of Biological Chemistry [Online] 284:22926-22937. Available at: http://dx.doi.org/10.1074/jbc.M109.019232.
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Amrinone is a bipyridine compound with characteristic effects on the force-velocity relationship of fast skeletal muscle, including a reduction in the maximum shortening velocity and increased maximum isometric force. Here we performed experiments to elucidate the molecular mechanisms for these effects, with the additional aim to gain insight into the molecular mechanisms underlying the force-velocity relationship. In vitro motility assays established that amrinone reduces the sliding velocity of heavy meromyosin-propelled actin filaments by 30% at different ionic strengths of the assay solution. Stopped-flow studies of myofibrils, heavy meromyosin and myosin subfragment 1, showed that the effects on sliding speed were not because of a reduced rate of ATP-induced actomyosin dissociation because the rate of this process was increased by amrinone. Moreover, optical tweezers studies could not detect any amrinone-induced changes in the working stroke length. In contrast, the ADP affinity of acto-heavy meromyosin was increased about 2-fold by 1 mm amrinone. Similar effects were not observed for acto-subfragment 1. Together with the other findings, this suggests that the amrinone-induced reduction in sliding velocity is attributed to inhibition of a strain-dependent ADP release step. Modeling results show that such an effect may account for the amrinone-induced changes of the force-velocity relationship. The data emphasize the importance of the rate of a strain-dependent ADP release step in influencing the maximum sliding velocity in fast skeletal muscle. The data also lead us to discuss the possible importance of cooperative interactions between the two myosin heads in muscle contraction.
Bloemink, M. et al. (2009). Alternative Exon 9-Encoded Relay Domains Affect More than One Communication Pathway in the Drosophila Myosin Head. Journal of Molecular Biology [Online] 389:707-721. Available at: http://dx.doi.org/10.1016/j.jmb.2009.04.036.
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We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide-binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced actomyosin dissociation rate constant (K(1)k(+2)) and ADP affinity (K(AD)), measured for the chimeric constructs IFI-9b and EMB-9a, compared to wild-type IFI and EMB values. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide-binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50-kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of actomyosin cross-bridge kinetics.
Smith, D. et al. (2008). Towards a Unified Theory of Muscle Contraction. I: Foundations. Annals of Biomedical Engineering [Online] 36:1624-1640. Available at: http://dx.doi.org/10.1007/s10439-008-9536-6.
Molecular models of contractility in striated muscle require an integrated description of the action of myosin motors, firstly in the filament lattice of the half-sarcomere. Existing models do not adequately reflect the biochemistry of the myosin motor and its sarcomeric environment. The biochemical actin-myosin-ATP cycle is reviewed, and we propose a model cycle with two 4- to 5-nm working strokes, where phosphate is released slowly after the first stroke. A smaller third stroke is associated with ATP-induced detachment from actin. A comprehensive model is defined by applying such a cycle to all myosin-S1 heads in the half-sarcomere, subject to generic constraints as follows: (a) all strain-dependent kinetics required for actin-myosin interactions are derived from reaction-energy landscapes and applied to dimeric myosin, (b) actin-myosin interactions in the half-sarcomere are controlled by matching rules derived from the structure of the filaments, so that each dimer may be associated with a target zone of three actin sites, and (c) the myosin and actin filaments are treated as elastically extensible. Numerical predictions for such a model are presented in the following paper.
Maytum, R. et al. (2008). Ultra short yeast tropomyosins show novel Myosin regulation. Journal of Biological Chemistry [Online] 283:1902-1910. Available at: http://dx.doi.org/10.1074/jbc.M708593200.
Tropomyosin (Tm) is an alpha-helical coiled-coil actin-binding protein present in all eukaryotes from yeast to man. Its functional role has been best described in muscle regulation; however its much wider role in cytoskeletal actin regulation is still to be clarified. Isoforms vary in size from 284 or 248 amino acids in vertebrates, to 199 and 161 amino acids in yeast, spanning from 7 to 4 actin binding sites respectively. In Saccharomyces cerevisiae, the larger yTm1 protein is produced by an internal 38-amino acid duplication, corresponding to a single actin-binding site. We have produced an ultra-short Tm with only 125 amino acids by removing both of the 38 amino acid repeats from yTm1, with the addition of an Ala-Ser extension used to mimic the essential N-terminal acetylation. This short Tm, and an M1T mutant of it, bind to actin with a similar affinity to most Tms previously studied (K(50%) approximately 0.5 mum). However, an equilibrium fluorescence binding assay shows a much greater inhibition of myosin binding to actin than any previously studied Tm. Actin cosedimentation assays show this is caused by direct competition for binding to actin. The M1T mutant shows a reduced inhibition, probably due to weaker end-to-end interactions making it easier for myosin to displace Tm. All previously characterized Tms, although able to sterically block the myosin-binding site, are able to bind to actin along with myosin. By showing that Tm can compete directly with myosin for the same binding site these new Tms provide direct evidence for the steric blocking model.
Adamek, N., Coluccio, L. and Geeves, M. (2008). Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear. Proceedings of the National Academy of Sciences of the United States of America. [Online] 105:5710-5715. Available at: http://dx.doi.org/10.1073/pnas.0710520105.
The class I myosin Myo1c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by Ca2+, permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a Myo1c fragment (motor domain and one IQ domain with associated calmodulin), with biochemical and kinetic properties similar to those of the native molecule, we have performed a thorough analysis of the biochemical cross-bridge cycle. We show that, although the steady-state ATPase activity shows little calcium sensitivity, individual molecular events of the cross-bridge cycle are calcium-sensitive. Of significance is a 7-fold inhibition of the ATP hydrolysis step and a 10-fold acceleration of ADP release in calcium. These changes result in an acceleration of detachment of the cross-bridge and a lengthening of the lifetime of the detached M-ATP state. These data support a model in which slipping adaptation, which reduces tip-link tension and allows the transduction channels to close after an excitatory stimulus, is mediated by Myo1c and modulated by the calcium transient.
Pearson, D., Swartz, D. and Geeves, M. (2008). Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W†. Biochemistry [Online] 47:12146-12158. Available at: http://dx.doi.org/10.1021/bi801150w.
We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.
Meshcheryakov, V. et al. (2008). Crystallization and preliminary X-ray crystallographic analysis of full-length yeast tropomyosin 2 from Saccharomyces cerevisiae. Acta Crystallogr Section F-Structural Biology and Crystallization Communications [Online] 64:528-530. Available at: http://dx.doi.org/10.1107/S1744309108013110.
Tropomyosin is a highly conserved actin-binding protein that is found in most eukaryotic cells. It is critical for actin-filament stabilization and for cooperative regulation of many actin functions. Detailed structural information on tropomyosin is very important in order to understand the mechanisms of its action. Whereas structures of isolated tropomyosin fragments have been obtained at high resolution, the atomic structure of the entire tropomyosin molecule is still unknown. Here, the crystallization and preliminary crystallographic analysis of full-length yeast tropomyosin 2 (yTm2) from Saccharomyces cerevisiae are reported. Recombinant yTm2 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 154.8, b = 49.9, c = 104.0 A, alpha = gamma = 90.0, beta = 124.0 degrees and two molecules in the asymmetric unit. A complete native X-ray diffraction data set was collected to 3.5 A resolution using synchrotron radiation.
Coulton, A. et al. (2008). Role of the Head-to-Tail Overlap Region in Smooth and Skeletal Muscle beta-Tropomyosin. Biochemistry [Online] 47:388-397. Available at: http://dx.doi.org/10.1021/bi701144g.
Tropomyosin (Tm) is an alpha-helical, parallel, two-chain coiled coil which binds along the length of actin filaments in both muscle and non-muscle cells. Smooth and skeletal muscle Tms differ extensively at the C-terminus encoded by exon 9. Replacement of the striated muscle specific exon 9a-encoded C-terminus with that encoded by exon 9d expressed in smooth muscle and non-muscle cells increases the affinity of unacetylated alpha-SkTm for actin [Cho, Y. J., and Hitchcock-Degregori, S. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10153-10157]. Here we show that swapping 10 amino acids at the C-terminus of beta-SkTm with the corresponding 10 amino acids of beta-SmTm had little effect on the regulation of S1 binding to actin, but Tm viscosity, Tm binding to actin, and troponin T1 binding to Tm all become like smooth rather than SkTm. beta-SkTm point mutations show that these properties are largely defined by the amino acids at two positions, 277 and 279. The N279L mutation reduces the viscosity of beta-SkTm to close to beta-SmTm values, while both residues contribute to the binding of TnT1. We also show that removing the first 11 N-terminal amino acids of beta-SmTm to make the mutant DeltaN-betaSmTm results in a 10-fold weakening in actin affinity compared to that of beta-SmTm. CD studies show no difference in thermal unfolding between beta-SmTm and DeltaN-betaSmTm; however, the viscosity of DeltaN-betaSmTm is much lower than that of the control. The results suggest that DeltaN-betaSmTm was unable to form filaments in solution but can form filaments on actin.
Bernstein, S. and Geeves, M. (2008). The XXXVII European muscle conference: Oxford September 2008. Journal of Muscle Research and Cell Motility [Online] 29:253-256. Available at: http://dx.doi.org/10.1007/s10974-008-9151-z.
Tsiavaliaris, G. et al. (2008). Mechanism, regulation, and functional properties of Dictyostelium myosin-1B. Journal of Biological Chemistry [Online] 283:4520-4527. Available at: http://dx.doi.org/10.1074/jbc.M708113200.
Myosin-1B is one of three long tailed class-1 myosins containing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes to actin-rich structures at the leading edge of migrating cells where it has been implicated in the formation and retraction of membrane projections, the recycling of plasma membrane components, and intracellular particle transport. Here, we have used a combination of molecular engineering approaches to describe the kinetic and motile properties of the myosin-1B motor and its regulation by TEDS site phosphorylation. Our results show that myosin-1B is a low duty ratio motor and displays the fastest nucleotide binding kinetics of any of the Dictyostelium class-1 myosins studied so far. Different from Dictyostelium myosin-1D and myosin-1E, dephosphorylated myosin-1B is not inactivated but moves actin filaments efficiently, albeit at an up to 8-fold slower velocity in the in vitro motility assay. A further difference is that myosin-1B lacks the ability to switch between rapid movement and bearing tension upon physiological changes of free Mg2+ ions. In this respect, its motor properties appear to be more closely related to Dictyostelium myosin-2 and rabbit skeletal muscle myosin.
Malnasi-Csizmadia, A. et al. (2007). Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release. Journal of Biological Chemistry [Online] 282:17658-17664. Available at: http://dx.doi.org/10.1074/jbc.M701447200.
After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.
Boussouf, S. et al. (2007). Role of tropomyosin isoforms in the calcium sensitivity of striated muscle thin filaments. Journal of Muscle Research and Cell Motility [Online] 28:49-58. Available at: http://www.springerlink.com/content/24jg471841626270/.
We have expressed alpha & beta isoforms of mammalian striated muscle tropomyosin (Tm) and alpha-Tm carrying the D175N or E180G cardiomyopathy mutations. In each case the Tm carries an Ala-Ser N-terminal extension to mimic the acetylation of the native Tm. We show that these Ala-Ser modified proteins are good analogues of the native Tm in the assays used here. We go on to use an in vitro kinetic approach to define the assembly of actin filaments with the Tm isoforms with either a cardiac or a skeletal muscle troponin (cTn, skTn). With skTn the calcium sensitivity of the actin filament is the same for alpha & beta-Tm and there is little change with the mutant Tms. For cTn switching from alpha to beta-Tm causes an increase of calcium sensitivity of 0.2 pCa units. D175N is very similar to the wild type alpha-Tm and E180G shows a small increase in calcium sensitivity of about 0.1 pCa unit. The formation of the switched-off blocked-state of the actin filament is independent of the Tm isoform but does differ for cardiac versus skeletal Tn. The in vitro assays developed here provide a novel, simple and efficient method for assaying the behaviour of expressed thin filament proteins.
Boussouf, S. et al. (2007). The regulation of Myosin binding to actin filaments by lethocerus troponin. Journal of Molecular Biology [Online] 373:587-98. Available at: http://dx.doi.org/10.1016/j.jmb.2007.07.066.
Lethocerus indirect flight muscle has two isoforms of troponin C, TnC-F1 and F2, which are unusual in having only a single C-terminal calcium binding site (site IV, isoform F1) or one C-terminal and one N-terminal site (sites IV and II, isoform F2). We show here that thin filaments assembled from rabbit actin and Lethocerus tropomyosin (Tm) and troponin (Tn) regulate the binding of rabbit myosin to rabbit actin in much the same way as the mammalian regulatory proteins. The removal of calcium reduces the rate constant for S1 binding to regulated actin about threefold, independent of which TmTn is used. This is consistent with calcium removal causing the TmTn to occupy the B or blocked state to about 70% of the total. The mid point pCa for the switch differed for TnC-F1 and F2 (pCa 6.9 and 6.0, respectively) consistent with the reported calcium affinities for the two TnCs. Equilibrium titration of S1 binding to regulated actin filaments confirms calcium regulated binding of S1 to actin and shows that in the absence of calcium the three actin filaments (TnC-F1, TnC-F2 and mammalian control) are almost indistinguishable in terms of occupancy of the B and C states of the filament. In the presence of calcium TnC-F2 is very similar to the control with approximately 80% of the filament in the C-state and 10-15% in the fully on M-State while TnC-F1 has almost 50% in each of the C and M states. This higher occupancy of the M-state for TnC-F1, which occurs above pCa 6.9, is consistent with this isoform being involved in the calcium activation of stretch activation. However, it leaves unanswered how a C-terminal calcium binding site of TnC can activate the thin filament.
Kintses, B. et al. (2007). Reversible movement of switch 1 loop of myosin determines actin interaction. EMBO Journal [Online] 26:265-274. Available at: http://www.nature.com/emboj/journal/v26/n1/pdf/7601482a.pdf.
The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin-myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in general
Geeves, M. et al. (2007). A variable domain near the ATP-binding site in Drosophila muscle myosin is part of the communication pathway between the nucleotide and actin-binding sites. Journal of Molecular Biology [Online] 368:1051-1066. Available at: http://dx.doi.org/10.1016/j.jmb.2007.02.042.
Drosophila expresses several muscle myosin isoforms from a single gene by alternatively splicing six of the 19 exons. Here we investigate exon 7, which codes for a region in the upper 50 kDa domain near the nucleotide-binding pocket. This region is of interest because it is also the place where a large insert is found in myosin VI and where several cardiomyopathy mutations have been identified in human cardiac myosin. We expressed and purified chimeric muscle myosins from Drosophila, each varying at exon 7. Two chimeras exchanged the entire exon 7 domain between the indirect flight muscle (IFI, normally containing exon 7d) and embryonic body wall muscle (EMB, normally containing exon 7a) isoforms to create IFI-7a and EMB-7d. The second two chimeras replaced each half of the exon 7a domain in EMB with the corresponding portion of exon 7d to create EMB-7a/7d and EMB-7d/7a. Transient kinetic studies of the motor domain from these myosin isoforms revealed changes in several kinetic parameters between the IFI or EMB isoforms and the chimeras. Of significance were changes in nucleotide binding, which differed in the presence and absence of actin, consistent with a model in which the exon 7 domain is part of the communication pathway between the nucleotide and actin-binding sites. Homology models of the structures suggest how the exon 7 domain might modulate this pathway.
Coulton, A. et al. (2007). Acetylation regulates tropomyosin function in the fission yeast Schizosaccharomyces pombe. Journal of Cell Science [Online] 120:1635-1645. Available at: http://dx.doi.org/10.1242/jcs.001115.
Tropomyosin is an evolutionarily conserved alpha-helical coiled-coil protein that promotes and maintains actin filaments. In yeast, Tropomyosin-stabilised filaments are used by molecular motors to transport cargoes or to generate motile forces by altering the dynamics of filament growth and shrinkage. The Schizosaccharomyces pombe tropomyosin Cdc8 localises to the cytokinetic actomyosin ring during mitosis and is absolutely required for its formation and function. We show that Cdc8 associates with actin filaments throughout the cell cycle and is subjected to post-translational modification that does not vary with cell cycle progression. At any given point in the cell cycle 80% of Cdc8 molecules are acetylated, which significantly enhances their affinity for actin. Reconstructions of electron microscopic images of actin-Cdc8 filaments establish that the majority of Cdc8 strands sit in the 'closed' position on actin filaments, suggesting a role in the regulation of myosin binding. We show that Cdc8 regulates the equilibrium binding of myosin to actin without affecting the rate of myosin binding. Unacetylated Cdc8 isoforms bind actin, but have a reduced ability to regulate myosin binding to actin. We conclude that although acetylation of Cdc8 is not essential, it provides a regulatory mechanism for modulating actin filament integrity and myosin function.
Bloemink, M. et al. (2007). Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle. Journal of Molecular Biology [Online] 373:1184-1197. Available at: http://dx.doi.org/10.1016/j.jmb.2007.08.050.
Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.
Iorga, B., Adamek, N. and Geeves, M. (2007). The slow skeletal muscle isoform of myosin shows kinetic features common to smooth and non-muscle myosins. Journal of Biological Chemistry [Online] 282:3559-3570. Available at: http://www.jbc.org/cgi/reprint/282/6/3559.
Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the,beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to similar to 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (similar to 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actinmyosin(.)ADP and provide evidence of three closely related actinmyosin(.)ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (similar to 20 s(-1)), and a third complex that releases ADP too slowly (similar to 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin(.)ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release
Adio, S. et al. (2006). Kinetic and mechanistic basis of the nonprocessive Kinesin-3 motor NcKin3. Journal of Biological Chemistry [Online] 281:37782-93. Available at: http://dx.doi.org/10.1074/jbc.M605061200.
Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 mum/s and correlates positively with motor density. The processivity index (k(bi,ratio)) of approximately 3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.
Herm-Gotz, A. et al. (2006). Functional and biophysical analyses of the class XIV Toxoplasma gondii Myosin D. Journal of Muscle Research and Cell Motility 27:139-51.
The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasite.
Coulton, A., Lehrer, S. and Geeves, M. (2006). Functional homodimers and heterodimers of recombinant smooth muscle tropomyosin. Biochemistry [Online] 45:12853-8. Available at: http://dx.doi.org/10.1021/bi0613224.
Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N-termini to bind strongly to actin. Tm containing an N-terminal alanine-serine (AS) extension to mimic acetylation has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both alpha alpha and beta beta smooth Tm homodimers. We show that (1) AS alpha-Tm binds actin 100-fold tighter than alpha-Tm and 2-fold tighter than native smooth alphabeta-Tm, (2) beta-Tm requires an AS extension to bind actin, and (3) AS beta-Tm binds actin 10-fold weaker than AS alpha-Tm. Tm is present in smooth muscle tissues as >95% heterodimer; therefore, we studied the binding of recombinant alphabeta heterodimers with different AS extensions. This study shows that recombinant Tm requires an AS extension on both alpha and beta chains to bind like native Tm and that the alpha chain contributes more to actin binding than the beta chain. Once assembled onto an actin filament, all smooth muscle Tm's regulate S1 binding to actin Tm in the same way, irrespective of the presence of an AS extension.
Rodger, A. et al. (2006). Looking at long molecules in solution: what happens when they are subjected to Couette flow? Physical Chemistry Chemical Physics [Online] 8:3161-71. Available at: http://dx.doi.org/10.1039/B604810M.
Knowing the structure of a molecule is one of the keys to deducing its function in a biological system. However, many biomacromolecules are not amenable to structural characterisation by the powerful techniques often used namely NMR and X-ray diffraction because they are too large, or too flexible or simply refuse to crystallize. Long molecules such as DNA and fibrous proteins are two such classes of molecule. In this article the extent to which flow linear dichroism (LD) can be used to characterise the structure and function of such molecules is reviewed. Consideration is given to the issues of fluid dynamics and light scattering by such large molecules. A range of applications of LD are reviewed including (i) fibrous proteins with particular attention being given to actin; (ii) a far from comprehensive discussion of the use of LD for DNA and DNA-ligand systems; (iii) LD for the kinetics of restriction digestion of circular supercoiled DNA; and (iv) carbon nanotubes to illustrate that LD can be used on any long molecules with accessible absorption transitions.
Durrwang, U. et al. (2006). Dictyostelium myosin-IE is a fast molecular motor involved in phagocytosis. Journal of Cell Science [Online] 119:550-58. Available at: http://dx.doi.org/10.1242/jcs.02774.
Class I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Here we describe the cellular localization of myosin-IE and its role in the phagocytic uptake of solid particles and cells. A complete analysis of the kinetic and motor properties of Dictyostelium discoideum myosin-IE was achieved by the use of motor domain constructs with artificial lever arms. Class I myosins belonging to subclass IC like myosin-IE are thought to be tuned for tension maintenance or stress sensing. In contrast to this prediction, our results show myosin-IE to be a fast motor. Myosin-IE motor activity is regulated by myosin heavy chain phosphorylation, which increases the coupling efficiency between the actin and nucleotide binding sites tenfold and the motile activity more than fivefold. Changes in the level of free Mg(2+) ions, which are within the physiological range, are shown to modulate the motor activity of myosin-IE by inhibiting the release of adenosine diphosphate.
Gewiss Mogensen, E. et al. (2006). Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1. Eukaryotic Cell [Online] 5:103-111. Available at: http://dx.doi.org/10.1128/EC.5.1.103-111.2006.
Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two beta-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH.
Nyitrai, M. et al. (2006). What Limits the Velocity of Fast-skeletal Muscle Contraction in Mammals? Journal of Molecular Biology [Online] 355:432-42. Available at: http://dx.doi.org/10.1016/j.jmb.2005.10.063.
In rat skeletal muscle the unloaded shortening velocity (V(o)) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit V(o). However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5M KCl, 22 degrees C for kinetic assays of myosin, 200mM ionic strength, 12 degrees C to measure V(o)). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, K(AD)) and the rate of ATP-induced acto-HMM dissociation (controlled by K(1)k(+2)) under the same experimental condition used to measure V(o). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit V(o), whereas K(1)k(+2) was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit V(o) for both isoforms.
Kremneva, E. et al. (2006). Thermal unfolding of smooth muscle and nonmuscle tropomyosin alpha-homodimers with alternatively spliced exons. FEBS Journal [Online] 273:588-600. Available at: http://dx.doi.org/10.1111/j.1742-4658.2005.05092.x.
We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.
Fujita-Becker, S. et al. (2005). Changes in Mg2+ ion concentration and heavy chain phosphorylation regulate the motor activity of a class I myosin. Journal of Biological Chemistry [Online] 280:6064-71. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15579903.
Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg(2+) ions on in vitro motility was discovered. Changes in the level of free Mg(2+) ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg(2+) ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension.
Clark, R. et al. (2005). Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access. Journal of Biological Chemistry [Online] 280:30935-42. Available at: http://www.jbc.org/cgi/content/abstract/280/35/30935.
Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.
Geeves, M., Fedorov, R. and Manstein, D. (2005). Molecular mechanism of actomyosin-based motility. Cellular and Molecular Life Sciences [Online] 62:1462-77. Available at: http://www.springerlink.com/content/j3q0w4gk15w58178/.
Sophisticated molecular genetic, biochemical and biophysical studies have been used to probe the molecular mechanism of actomyosin-based motility. Recent solution measurements, high-resolution structures of recombinant myosin motor domains, and lower resolution structures of the complex formed by filamentous actin and the myosin motor domain provide detailed insights into the mechanism of chemomechanical coupling in the actomyosin system. They show how small conformational changes are amplified by a lever-arm mechanism to a working stroke of several nanometres, explain the mechanism that governs the directionality of actin-based movement, and reveal a communication pathway between the nucleotide binding pocket and the actin-binding region that explains the reciprocal relationship between actin and nucleotide affinity. Here we focus on the interacting elements in the actomyosin system and the communication pathways in the myosin motor domain that respond to actin binding.
Nalavadi, V. et al. (2005). Kinetic Mechanism of Myosin IXB and the Contributions of Two Class IX-specific Regions. Journal of Biological Chemistry [Online] 280:38957-38968. Available at: http://www.jbc.org/cgi/content/abstract/280/47/38957.
Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.
Geeves, M. and Holmes, K. (2005). The molecular mechanism of muscle contraction. Fibrous Proteins: Muscle and Molecular Motors [Online] 71:161-193. Available at: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B7CTK-4HC15BD-5&_user=125871&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000010239&_version=1&_urlVersion=0&_userid=125871&md5=461c86721444b98b516e70b4c96e6fcc.
Shimada, A. et al. (2004). The core FH2 domain of diaphanous-related formins is an elongated actin binding protein that inhibits polymerization. Molecular Cell [Online] 13:511-522. Available at: http://dx.doi.org/10.1016/S1097-2765(04)00059-0.
Diaphanous-related formins (Drf) are activated by Rho GTP binding proteins and induce polymerization of unbranched actin filaments. They contain three formin homology domains. Evidence as to the effect of formins on actin polymerization were obtained using FH2/FH1 constructs of various length from different Drfs. Here we define the core FH2 domain as a proteolytically stable domain of approximately 338 residues. The monomeric FH2 domains from mDia1 and mDia3 inhibit polymerization of actin and can bind in a 1:1 complex with F-actin at micromolar concentrations. The X-ray structure analysis of the domain shows an elongated, crescent-shaped molecule consisting of three helical subdomains. The most highly conserved regions of the domain span a distance of 75 A and are both required for barbed-end inhibition. A construct containing an additional 72 residue linker has dramatically different properties: It oligomerizes and induces actin polymerization at subnanomolar concentration.
Kremneva, E. et al. (2004). Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin. Biophysical Journal [Online] 87:3922-33. Available at: http://dx.doi.org/10.1529/biophysj.104.048793.
Differential scanning calorimetry was used to investigate the thermal unfolding of native alpha-tropomyosin (Tm), wild-type alpha-Tm expressed in Escherichia coli and the wild-type alpha-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G. Recombinant alpha-Tm was expressed with an N-terminal Ala-Ser extension to substitute for the essential N-terminal acetylation of the native Tm. Native and Ala-Ser-Tm were indistinguishable in our assays. In the absence of F-actin, the thermal unfolding of Tm was reversible and the heat sorption curve of Tm with Cys-190 reduced was decomposed into two separate calorimetric domains with maxima at approximately 42 and 51 degrees C. In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47 degrees C and completely disappears after the irreversible denaturation of F-actin. A good correlation was found to exist between the maximum of this peak and the temperature of half-maximal dissociation of the F-actin/Tm complex as determined by light scattering experiments. We conclude that Tm thermal denaturation only occurs upon its dissociation from F-actin. In the presence of F-actin, D175N alpha-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type alpha-Tm. The actin-induced stabilization of E180G alpha-Tm is significantly less than for wild-type alpha-Tm and D175N alpha-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation.
Maytum, R. et al. (2004). Tropomyosin exon 6b is troponin-specific and required for correct acto-myosin regulation. Journal of Biological Chemistry [Online] 279:18203-9. Available at: http://dx.doi.org/10.1074/jbc.M311636200.
The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT(1)-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 microm) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin.Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin.Tm.Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin.Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.
Crevel, I. et al. (2004). What kinesin does at roadblocks: the coordination mechanism for molecular walking. EMBO Journal [Online] 23:23-32. Available at: http://dx.doi.org/10.1038/sj.emboj.7600042.
Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s(-1). Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50-70 s(-1)), indicating that during walking, lead head attachment induces at most only a slight (less than two-fold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.
Nyitrai, M. and Geeves, M. (2004). Adenosine diphosphate and strain sensitivity in myosin motors. Philosophical Transactions of the Royal Society B: Biological Sciences [Online] 359:1867-77. Available at: http://dx.doi.org/10.1098%2Frstb.2004.1560.
The release of adenosine diphosphate (ADP) from the actomyosin cross-bridge plays an important role in the adenosine-triphosphate-driven cross-bridge cycle. In fast contracting muscle fibres, the rate at which ADP is released from the cross-bridge correlates with the maximum shortening velocity of the muscle fibre, and in some models the rate of ADP release defines the maximum shortening velocity. In addition, it has long been thought that the rate of ADP release could be sensitive to the load on the cross-bridge and thereby provide a molecular explanation of the Fenn effect. However, direct evidence of a strain-sensitive ADP-release mechanism has been hard to come by for fast muscle myosins. The recently published evidence for a strain-sensing mechanism involving ADP release for slower muscle myosins, and in particular non-muscle myosins, is more compelling and can provide the mechanism of processivity for motors such as myosin V. It is therefore timely to examine the evidence for this strain-sensing mechanism. The evidence presented here will argue that a strain-sensitive mechanism of ADP release is universal for all myosins but the basic mechanism has evolved in different ways for different types of myosin. Furthermore, this strain-sensing mechanism provides a way of coordinating the action of multiple myosin motor domains in a single myosin molecule, or in complex assemblies of myosins over long distances without invoking a classic direct allosteric or cooperative communication between motors.
Attwood, P. and Geeves, M. (2004). Kinetics of an enzyme-catalyzed reaction measured by electrospray ionization mass spectrometry using a simple rapid mixing attachment. Analytical Biochemistry [Online] 334:382-9. Available at: http://dx.doi.org/10.1016/j.ab.2004.08.010.
Mass spectrometry offers a potential means of measuring virtually all enzyme-catalyzed reactions by simultaneously measuring the concentrations of substrates, products, and intermediates where there are differences in mass between them. To perform these measurements the reaction mixture must be aged for different times and then ionized. Electrospray ionization mass spectrometry provides the most direct means of measuring these reactions. Here we describe a simple reaction mixing and ageing attachment for an electrospray ionization mass spectrometer, built from commercially available components. We have employed this device to measure the kinetics of a model reaction, namely the hydrolysis of N2-(carbobenzyloxy)-L-lysine-p-nitrophenyl ester-catalyzed by trypsin. In this way we were able to measure the kinetics of substrate depletion, product formation, and changes in both free enzyme and acyl-enzyme intermediate concentration in the approach to steady state. With this device we were able to measure reaction times down to about 640 ms.
Batters, C. et al. (2004). Myo1c is designed for the adaptation response in the inner ear. Embo Journal [Online] 23:1433-40. Available at: http://dx.doi.org/ 10.1038/sj.emboj.7600169.
The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.
Smith, D. and Geeves, M. (2003). Cooperative regulation of myosin-actin interactions by a continuous flexible chain II: actin-tropomyosin-troponin and regulation by calcium. Biophysical Journal [Online] 84:3168-3180. Available at: http://dx.doi.org/10.1016/S0006-3495(03)70041-1.
The model of myosin regulation by a continuous tropomyosin chain is generalized to a chain of tropomyosin-troponin units. Myosin binding to regulated actin is cooperative and initially inhibited by the chain as before. In the absence of calcium, myosin is further inhibited by the binding of troponin-I to actin, which through the whole of troponin pins the tropomyosin chain in a blocking position; myosin and TnI compete for actin and induce oppositely-directed chain kinks. The model predicts equilibrium binding curves for myosin-S1 and TnI as a function of their first-order affinities K(S1) and L(TI). Myosin is detached by the actin binding of TnI, but TnI is more efficiently detached by myosin when the kink size (typically nine to ten actin sites) spans the seven-site spacing between adjacent TnI molecules. An allosteric mechanism is used for coupling the detachment of TnI to calcium binding by TnC. With thermally activated TnI kinks (kink energy B approximately k(B)T), TnI also binds cooperatively to actin, producing cooperative detachment of myosin and biphasic myosin-calcium Hill plots, with Hill coefficients of 2 at high calcium and 4-6 at low calcium as observed in striated muscle. The theory also predicts the cooperative effects observed in the calcium loading of TnC.
Silva, R., Sparrow, J. and Geeves, M. (2003). Isolation and kinetic characterisation of myosin and myosin S1 from the Drosophila indirect flight muscles. Journal of Muscle Research and Cell Motility [Online] 24:489-498. Available at: http://dx.doi.org/10.1023/B:JURE.0000009809.69829.74.
The potential to explore myosin function through the alternative exons and mutations of the single muscle myosin heavy chain gene, Mhc of Drosophila requires detailed kinetic analysis of the myosins. We have obtained microgram quantities of enzymatically active Drosophila myosin and subfragment 1 (S1) from dissected indirect flight muscles. Using recent developments in stopped-flow and flash-photolysis methods combined with fluorescent/light scattering technologies we have determined some of the key kinetic parameters of actin-myosin and myosin-nucleotide interactions. The rate of ATP-induced dissociation of actin from Drosophila myosin (0.23 microM(-1) s(-1)) and subfragment 1 (S1, 0.82 microM(-1) s(-1)) are both fast and similar to values measured for mammalian skeletal muscle myosins and S1 fragments respectively. The ATP-induced cross bridge dissociation of Drosophila acto.S1 is expected to be fast since, for a rapidly contracting muscle like the Drosophila flight muscle, the post power stroke cross bridge must detach rapidly from actin or become a drag on the contracting filament. ATP-induced detachment is preceded by ADP release and this is proposed as the rate-limiting step that defines muscle shortening velocity. We show that the affinity of ADP for acto.S1 at 400 microM is 2-3 fold weaker than fast vertebrate myosins. This leads to an estimate of the ADP release rate constant of 4000 s(-1). We show that this predicts a maximum shortening velocity very similar to that obtained from in vivo estimates of indirect flight muscle shortening. The data is therefore compatible with ADP dissociation limiting the in vivo shortening velocity.
Maytum, R. et al. (2003). Differential regulation of the actomyosin interaction by skeletal and cardiac troponin isoforms. Journal of Biological Chemistry [Online] 278:6696-6701. Available at: http://dx.doi.org/10.1074/jbc.M210690200.
There are significant isoform differences between the skeletal and cardiac troponin complexes. Studies of the regulatory properties of these proteins have previously shown only significant differences in the calcium dependence of their regulation. Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves. However in the absence of calcium the S1 binding curves differ considerably. Combined with kinetic measurements, curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in K(T) (the closed-open equilibrium) for the skeletal system but little change in the cardiac system. The results show a significant difference in the range of regulatory effect between the cardiac and skeletal systems that we interpret as effects upon actin-troponin (Tn)I-TnC binding equilibria. As structural data show that the Ca(2+)-bound TnC structures differ, the additional counter-intuitive result here is that with respect to myosin binding the +Ca(2+) state of the two systems is similar whereas the -Ca(2+) state differs. This shows the regulatory tuning of the troponin complex produced by isoform variation is the net result of a complex series of interactions among all the troponin components.
Clark, R. et al. (2003). Probing nucleotide dissociation from myosin in vitro using microgram quantities of myosin. Journal of Muscle Research and Cell Motility [Online] 24:315-321. Available at: http://dx.doi.org/10.1023/A:1025481908301.
The detailed kinetic analysis of novel myosin motors is often limited by the quantity of stable protein available for study. We show here that the use of coumarin based fluorescent ADP analogues allows the assay of ADP affinities and dissociation rate constants in a flash photolysis apparatus using microg quantities of the rabbit muscle myosin S1. We go on to use the analogues to characterise two other rat muscle myosin S1 and the motor domain of Dictyostelium cytoplasmic myosin II. The results show that the fluorescence change for the binding of a coumarin based ADP analogue to a myosin motor domain is variable in sign as well as amplitude for the different proteins. The analysis also provided estimates of the affinities of caged-ATP for S1 which were < or = 10 microM for muscle S1s and > 200 microM for the non-muscle myosin.
Smith, D., Maytum, R. and Geeves, M. (2003). Cooperative regulation of myosin-actin interactions by a continuous flexible chain I: actin-tropomyosin systems. Biophysical Journal [Online] 84:3155-3167. Available at: http://dx.doi.org/10.1016/S0006-3495(03)70040-X.
We present a model for cooperative myosin binding to the regulated actin filament, where tropomyosins are treated as a weakly-confined continuous flexible chain covering myosin binding sites. Thermal fluctuations in chain orientation are initially required for myosin binding, leaving kinked regions under which subsequent myosins may bind without further distortion of the chain. Statistical mechanics predicts the fraction of sites with bound myosin-S1 as a function of their affinities. Published S1 binding curves to regulated filaments with different tropomyosin isoforms are fitted by varying the binding constant, chain persistence length nu (in actin monomers), and chain kink energy A from a single bound S1. With skeletal tropomyosin, we find an S1 actin-binding constant of 2.2 x 10(7) M(-1), A = 1.6 k(B)T and nu = 2.7. Similar persistence lengths are found with yeast tropomyosin. Larger values are found for tropomyosin-troponin in the presence of calcium (nu = 3.7) and tropomyosins from smooth muscle and fibroblasts (nu = 4.5). The relationship of these results to structural information and the rigid-unit model of McKillop and Geeves is discussed.
Miller, B. et al. (2003). Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling. Journal of Biological Chemistry [Online] 278:50293-50300. Available at: http://dx.doi.org/10.1074/jbc.M308318200.
The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by alpha-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin.myosin.ADP states may define actin filament velocity for these myosin isoforms.
Nyitrai, M., Szent-Gyorgyi, A. and Geeves, M. (2003). Interactions of the two heads of scallop (Argopecten irradians) heavy meromyosin with actin: influence of calcium and nucleotides. Biochemical Journal [Online] 370:839-848. Available at: http://dx.doi.org/10.1042/BJ20021519.
We recently proposed a co-operative model for the influence of calcium and ADP on scallop ( Argopecten irradians ) muscle heavy meromyosin (scHMM), in which scHMM exists in two conformations (designated 'off' and 'on'), and calcium and ADP are allosteric effectors of the equilibrium between the off and on conformations [Nyitrai, Szent-Gyorgyi and Geeves (2002) Biochem. J. 365, 19-30]. Here we examine the influence of actin on scHMM. In the absence of nucleotide, both heads of scHMM bind very tightly to actin, independent of the presence of calcium. In the absence of calcium, ADP dissociates scHMM from actin completely, and little evidence of ternary complex formation can be found (actin affinity >20 microM). The off state of scHMM therefore does not interact with actin. In the presence of calcium, ADP and actin lower each other's affinity for scHMM by 30-50-fold, although both heads remain strongly attached to actin (actin affinity 0.17 microM). Detailed analysis suggests that the second head contributes far more to the overall binding energy than is the case for mammalian skeletal muscle HMM. This is consistent with a different stereochemical relationship between the two heads in scallop and mammalian HMM molecules.
Nyitrai, M. et al. (2003). Ionic interactions play a role in the regulatory mechanism of scallop heavy meromyosin. Biophysical Journal [Online] 85:1053-62. Available at: http://dx.doi.org/10.1016/S0006-3495(03)74544-5.
Heavy meromyosin from scallop (scHMM) striated muscle is regulated by calcium binding to the essential light chain. The regulation can be modeled with a calcium-dependent equilibrium between on and off scHMM conformations. The observed rate constant for mant-ADP dissociation from scHMM is calcium dependent, and we show here that it can be used to define the equilibrium constant (K(eq)) between on and off conformations. The data show that K(eq) is markedly ionic strength dependent, with high salt (>/=200 mM) abolishing the off state even in the absence of calcium and low salt (<50 mM) favoring the off state even in the presence of calcium. Debye-Huckel plots of the equilibrium constant (K(eq)) for the on and off forms gave parallel slopes (5.94 +/- 0.33 and 6.36 +/- 0.17 M(-0.5)) in the presence and absence of calcium. The presence of an equilibrium mixture of two conformations was confirmed by sedimentation data and the effects of ADP, calcium and ionic strength were in qualitative agreement. Thus scHMM exists in two conformations that can be distinguished in sedimentation profiles and by the rate of release of mant-ADP. Increasing salt concentrations biases the system toward the on state, suggesting a role for ionic interactions in stabilizing the off state.
Maytum, R., Geeves, M. and Lehrer, S. (2002). A modulatory role for the troponin T tail domain in thin filament regulation. Journal of Biological Chemistry [Online] 277:29774-29780. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12045197.
In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI- TnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT(1) tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT(1) producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C- to M-state equilibrium, K(T), for TnT(1). This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.
Attwood, P. and Geeves, M. (2002). Changes in catalytic activity and association state of pyruvate carboxylase which are dependent on enzyme concentration. Archives of Biochemistry and Biophysics [Online] 401:63-72. Available at: http://dx.doi.org/10.1016/S0003-9861(02)00039-5.
The specific activity of chicken liver pyruvate carboxylase has been shown to decrease with decreasing enzyme concentration, even at 100 microM, which is close to the estimated physiological concentration. The kinetics of the loss of enzyme specific activity following dilution were biphasic. Incubation of dilution-inactivated enzyme with ATP, acetyl CoA, Mg2+ + ATP or, to a lesser degree, with Mg2+ alone resulted in a high degree of reactivation, while no reactivation occurred in the presence of pyruvate. The association state of the enzyme before, during, and after dilution inactivation has been assessed by gel filtration chromatography. These studies indicate that on dilution, there is dissociation of the catalytically active tetrameric enzyme species into inactive dimers. Reactivation of the enzyme resulted in reassociation of enzymic dimers into tetramers. The enzyme was shown to form high molecular weight aggregates at high enzyme concentrations.
Nyitrai, M., Szent-Gyorgyi, A. and Geeves, M. (2002). A kinetic model of the co-operative binding of calcium and ADP to scallop (Argopecten irradians) heavy meromyosin. Biochemical Journal [Online] 365:19-30. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12071838.
Analysis of the kinetics of ATP and ADP binding to scallop (Argopecten irradians) heavy meromyosin (HMM) showed that the only calcium-dependent process is the rate of ADP release. At physiological ionic strength calcium accelerated ADP release about 20-fold. Notably in the absence of calcium only one ADP bound HMM, with an affinity of 0.5-1 microM. The second nucleotide site remained unoccupied at up to 50 microM ADP yet could bind ATP rapidly. The calcium dependence of ADP-release rates showed that calcium binds co-operatively to scallop HMM with an affinity of 0.78 microM and a Hill coefficient of 1.9. Detailed interpretation of the data suggests that HMM exists in equilibrium between the on and off states and that calcium and ADP modulate the equilibrium between the two states. The on state is favoured in the presence of calcium and in the absence of both calcium and nucleotide. The off state is favoured by ADP (or ADP * P(i)) in the absence of calcium. A detailed co-operative model of the interaction of ADP and calcium with HMM is presented.
Geeves, M. and Lehrer, S. (2002). Modeling thin filament cooperativity. Biophysical Journal [Online] 82:1677-1679. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11898795.
Herm-Gotz, A. et al. (2002). Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor. EMBO Journal [Online] 21:2149-2158. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11980712.
Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding.
Pearson, D. et al. (2002). A novel pressure-jump apparatus for the microvolume analysis of protein-ligand and protein-protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1. Biochemical Journal [Online] 366:643-651. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12010120.
Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50 microl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383-1393]. This equipment can apply pressure changes of +/-20 MPa (maximally) in time periods as short as 80 micros and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1 min) exchange of samples. In the present study, we show that this system can perturb the binding of 2'(3')-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2'(3')-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23 degrees tilt in the tail of S1 bound to actin.
Geeves, M. (2002). Stretching the lever-arm theory. Nature [Online] 415:129-131. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11805818.
Tsiavaliaris, G. et al. (2002). Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium. EMBO Reports [Online] 3:1099-1105. Available at: http://dx.doi.org/10.1093/embo-reports/kvf214.
Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an approximately 100-fold increase in the mutants' affinity for actin in the presence of ATP.
Malnasi-Csizmadia, A. et al. (2001). Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue. Biochemistry [Online] 40:12727-12737. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11601998.
The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.
Rogers, K. et al. (2001). KIF1D is a fast non-processive kinesin that demonstrates novel K-loop-dependent mechanochemistry. Embo Journal [Online] 20:5101-5113. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11566875.
The KIF1 subfamily members are monomeric and contain a number of amino acid inserts in surface loops. A particularly striking insertion of several lysine/arginine residues occurs in L12 and is called the K-loop. Two recent studies have employed both kinetic and single-molecule methods to investigate KIF1 motor properties and have produced very different conclusions about how these motors generate motility. Here we show that a hitherto unstudied member of this group, KIF1D, is not chemically processive and drives fast motility despite demonstrating a slow ATPase. The K-loop of KIF1D was analysed by deletion and insertion mutagenesis coupled with characterization by steady state and transient kinetics. Together, the results indicate that the K-loop not only increases the affinity of the motor for the MT, but crucially also inhibits its subsequent isomerization from weak to strong binding, with coupled ADP release. By stabilizing the weak binding, the K-loop establishes a pool of motors primed to undergo their power stroke.
Lohmann, K. et al. (2001). Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization. Protein Expression and Purification [Online] 21:49-59. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11162386.
All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.
Maytum, R. et al. (2001). Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence. Biochemistry [Online] 40:7334-7341. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11401582.
The regulatory properties of naturally occurring tropomyosins (Tms) of differing lengths have been examined. These Tms span from 4 to 7 actin subunits. Native proteins have been used to study the common 7 actin-spanning skeletal and smooth muscle variants and expressed recombinant proteins to study the shorter fibroblast 5a, 5b, yeast Tm1 and yeast Tm2 Tms (6, 6, 5, and 4 actin-spanning variants, respectively). The yTm2 has been overexpressed in Escherichia coli with N-terminal constructs equivalent to those previously used for yTm1 [Maytum, R., et al. (2000) Biochemistry 39, 11913]. The regulation of myosin subfragment 1 (S1) binding to actin by Tm has been assessed using a sensitive S1 binding titration. The equilibrium between closed and open (C to M states, KT = 0.1-0.14) was similar for all vertebrate Tms. Apart from skTm where the apparent cooperative unit size (n) is the same as the structural size (n = 7 actin sites), the other vertebrate Tms that were studied exhibited large n values (n = 12-14). The yeast Tms also exhibited large values of n (6-9) in comparison to their structural sizes (4-5). The determined value of KT depended on the N-terminal sequence (KT = 0.15-1). These results are compared with the effect of S1 upon Tm's affinity for actin. The yeast Tms have regulatory parameters similar to those of skTm, but unlike skTm, S1 has little effect upon their actin affinity. This shows that an actin state with a high affinity for S1 and Tm is not necessary for regulation, and the higher affinity of S1 for actin in the presence of vertebrate Tms is probably the result of a direct interaction of S1 with Tm.
Berger, C. et al. (2001). ADP binding induces an asymmetry between the heads of unphosphorylated myosin. Journal of Biological Chemistry [Online] 276:23240-23245. Available at: http://dx.doi.org/10.1074/jcb.M100524200.
Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.
Weiss, S. et al. (2001). Differing ADP Release Rates from Myosin Heavy Chain Isoforms Define the Shortening Velocity of Skeletal Muscle Fibers. Journal of Biological Chemistry 276:45902-45908.
To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 microg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (K(AD)). The apparent second order rate constant of actomyosin dissociation gets faster (K(1)k(+2) = 0.17 -0.26 microm(-1) x s(-1)), whereas the affinity for ADP is weakened (250-930 microm) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V(0)) of the parent fibers. If the value of K(AD) is controlled largely by the rate constant of ADP release (k(-AD)), then the estimated value of k(-AD) is sufficiently low to limit V(0). In contrast, [ATP]K(1)k(+2) at a physiological concentration of 5 mm ATP would be 2.5-6 times faster than k(-AD).
Furch, M., Geeves, M. and Manstein, D. (2000). Kinetic dissection of the effect of negative charges of loop 2 on the enzymatic activity of myosin. Biophysical Journal 78:393-393.
Holmes, K. and Geeves, M. (2000). The structural basis of muscle contraction. Philosophical Transactions of the Royal Society B: Biological Sciences [Online] 355:419-431. Available at: http://dx.doi.org/10.1098/rstb.2000.0583.
The myosin cross-bridge exists in two conformations, which differ in the orientation of a long lever arm. Since the lever arm undergoes a 60 degrees rotation between the two conformations, which would lead to a displacement of the myosin filament of about 11 nm, the transition between these two states has been associated with the elementary 'power stroke' of muscle. Moreover, this rotation is coupled with changes in the active site (CLOSED to OPEN), which probably enable phosphate release. The transition CLOSED to OPEN appears to be brought about by actin binding. However, kinetics shows that the binding of myosin to actin is a two-step process which affects both ATP and ADP affinity and trice versa. The structural basis of these effects is only partially explained by the presently known conformers of myosin. Therefore, additional states of the myosin cross-bridge should exist. Indeed, cryoelectron microscopy has revealed other angles of the lever arm induced by ADP binding to a smooth muscle actin-myosin complex.
Lehman, W. et al. (2000). Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments. Journal of Molecular Biology [Online] 302:593-606. Available at: http://dx.doi.org/10.1006/jmbi.2000.4080.
Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps. one induced by Ca2+ binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and nonmuscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.
Coluccio, L. et al. (2000). Truncation of a mammalian myosin I results in loss of Ca2+-sensitive motility. Journal of Biological Chemistry [Online] 275:21618-21623. Available at: http://dx.doi.org/10.1074/jbc.M000363200.
MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4–6 associated calmodulins, is represented by the 130-kDa myosin I (or MI130) from rat liver. MI130 translocates actin filaments in vitro in a Ca2+-regulated manner. A decrease in motility observed at higher Ca2+ concentrations has been attributed to calmodulin dissociation. To investigate mammalian myosin I regulation, we have coexpressed in baculovirus calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic "motor" domain and the first calmodulin-binding IQ domain of ratmyr-1; we refer to this truncated molecule here as MI1IQ. Association of calmodulin to MI1IQ is Ca2+-insensitive. MI1IQ translocates actin filaments in vitro at a rate resembling MI130, but unlike MI130, does not exhibit sensitivity to 0.1–100 μm Ca2+. In addition to demonstrating successful expression of a functional truncated mammalian myosin Iin vitro, these results indicate that: 1) Ca2+-induced calmodulin dissociation from MI130in the presence of actin is not from the first IQ domain, 2) velocity is not affected by the length of the IQ region, and 3) the Ca2+ sensitivity of actin translocation exhibited by MI130 involves 1 or more of the other 5 IQ domains and/or the carboxyl tail.
Geeves, M., Perreault-Micale, C. and Coluccio, L. (2000). Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding. Journal of Biological Chemistry [Online] 275:21624-21630. Available at: http://dx.doi.org/10.1074/jbc.M000342200.
MI1IQ is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI130). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI1IQ. Transient kinetics reveal that the affinity of MI1IQ for actin is 12 nM. The ATP-induced dissociation of actin-MI1IQ is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca2+ sensitivity. The fast phase is eliminated by the addition of ADP, 10 mu M being required for half-saturation of the effect in the presence of Ca2+ and 3 mu M ADP in the absence of Ca2+. The slow phase shares the same rate constant as ADP release (8 and 3 s(-1) in the presence and absence of Ca2+, respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI130 and that the Ca2+ sensitivity of the kinetics are in all likelihood due to Ca2+ binding to the first IQ domain.
Maytum, R., Geeves, M. and Konrad, M. (2000). Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification. Biochemistry [Online] 39:11913-11920. Available at: http://dx.doi.org/10.1021/bi000977g.
The yeast tropomyosin 1 gene (TPM1) encodes the major isoform of the two tropomyosins (Tm) found in yeast. The gene has been expressed in E. coli and the protein purified. The gene product (yTm1) is a 199-amino acid protein that has a low affinity for actin compared to the native yTm1 purified from yeast. Mass spectrometry shows that the native protein is acetylated while the recombinant protein is not. A series of yTm1 N-terminal constructs were made with either an Ala-Ser dipeptide extension previously shown to restore actin binding to skeletal muscle Tm or the natural extension found in fibroblast Tm 5a/b. All constructs bound actin tightly and showed similar CD spectra and thermal stability. All constructs induced cooperativity in the equilibrium binding of myosin subfragment 1, to actin but the binding curves differed significantly between the constructs. The apparent cooperative unit size (n) and closed/open equilibrium (K-T) were determined using a fluorescence titration technique [Maytum et al. (1998) Biophys, J. 74, A347]. The data could be accounted for by changes in K-T (0.1-1) With no change in n, Values of n were approximately twice the structural unit size (5 actin sites). The presence of yTm on actin had little effect upon the overall affinity of S1 for actin despite showing an ability to regulate the acto-myosin interaction, These results show that the short yTm can aid our understanding of actomyosin regulation and that the N-terminus of Tm has a major influence upon its regulatory properties.
Weiss, S. et al. (2000). Kinetic characterisation of the myosin A from toxoplasma gondii. Biophysical Journal 78:1432Pos.
Furch, M. et al. (2000). Stabilization of the actomyosin complex by negative charges on myosin. Biochemistry [Online] 39:11602-11608. Available at: http://dx.doi.org/10.1021/bi000985x.
Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideum myosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemisty 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K-A) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K-A Stem almost exclusively from variations in: the dissociation rate constant (k(-A)), with the introduction of a single negative charge at position 532 having the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region.
Maytum, R., Geeves, M. and Lehrer, S. (2000). Effects of TnT and TnT1 upon regulation of actin-tropomyosin. Biophysical Journal 78:365A-365A.
Geeves, M., Chai, M. and Lehrer, S. (2000). Inhibition of actin-myosin subfragment 1 ATPase activity by troponin I and IC: Relationship to the thin filament states of muscle. Biochemistry [Online] 39:9345-9350. Available at: http://dx.doi.org/10.1021/bi0002232.
Troponin I (TnI) is the component of the troponin complex that inhibits actomyosin ATPase activity, and Ca2+ binding to the troponin C (TnC) component reverses the inhibition. Effects of the binding of TnI and the TnI-TnC (TnIC) complex to actin-tropomyosin (actinTm) on ATPase and on the binding kinetics of myosin subfragment 1 (S1) were studied to clarify the mechanism of the inhibition. TnI and TnIC in the absence of Ca2+ bind to actinTm and inhibit ATPase to similar levels with a stoichiometry of one TnI or one TnIC per one Tm and seven actin subunits. Tnf also binds to actinTmTn in the presence of Ca2+ with a stoichiometry and inhibition constant similar to those for the binding to actinTm of TnI and Tn in the absence of Ca2+. Thus, in the presence of Ca2+, the intrinsic TnI which is released from its binding site on actinTm does not interfere with the binding of an extra molecule of TnI to actinTmTn. The rate of Si binding to actinTmTnI and to actinTmTnTnI in the presence of Ca2+ was inhibited to the same extent as upon removal of Ca2+ from actinTmTn. These studies show that TnI inhibits ATPase by the same mechanism as Tn in the absence of Ca2+, by shifting the thin filament equilibria from the open state to the closed and blocked states.
Weiss, S., Chizhov, I. and Geeves, M. (2000). A flash photolysis fluorescence/light scattering apparatus for use with sub microgram quantities of muscle proteins. Journal of Muscle Research and Cell Motility [Online] 21:423-432. Available at: http://dx.doi.org/10.1023/A:1005690106951.
Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However, these methods traditionally require relatively large amounts of protein (10(-3) g) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g., Dictyostelium). We have investigated the use of flash photolysis of an inert precursor of ATP (cATP) to initiate the dissociation of acto.S1 and acto.myosin and the subsequent ATP turnover reaction. Using a sample volume of 10 mul we show that a significant amount of information on the transient and steady-state kinetics of the system can be obtained from a sample containing just 50 nM of acto.myosin or acto.S1 complex in solution. Therefore in presence of excess of one protein component the measurements require only 250 ng myosin, 62 ng S1 or 25 ng actin. This is therefore the method of choice for kinetic analysis of acto.myosins which are only available in microgram quantities. We report for the first time the determination of the second order rate constant of ATP-induced dissociation of actin from the myosin extracted from a single fibre from a rabbit psoas muscle.
Book section
Toseland, C. and Geeves, M. (2014). Rapid reaction kinetic techniques. in: Toseland, C. P. and Fili, N. eds. Fluorescent Methods Applied to Molecular Motors: from single molecules to whole cells. Springer, pp. 49-64. Available at: http://dx.doi.org/10.1007/978-3-0348-0856-9.
Adamek, N. and Geeves, M. (2014). Use of pyrene-labelled actin to probe actin-Myosin interactions: kinetic and equilibrium studies. in: Fluorescent Methods Applied to Molecular Motors: from single molecules to whole cells. Springer, pp. 87-104. Available at: http://link.springer.com/book/10.1007%2F978-3-0348-0856-9.
Johnson, M., Geeves, M. and Mulvihill, D. (2013). Production of Amino-Terminally Acetylated Recombinant Proteins in E. coli. in: Hake, S. and Janzen, C. eds. Protein Acetylation. Humana Press, pp. 193-200. Available at: http://dx.doi.org/10.1007/978-1-62703-305-3_15.
The majority of proteins in eukaryote cells are subjected to amino-terminal acetylation. This co-translational modification can affect the stability of a protein and also regulate its biological function. Amino-terminally acetylated recombinant proteins cannot be produced using prokaryote expression systems, such as E. coli, as these cells lack the appropriate N-α-terminal acetyltransferase complexes. Here we describe a simple protocol that allows the recombinant expression and purification of NatB-dependent amino-terminally acetylated proteins from E. coli.
Geeves, M. and Pearson, D. (2013). Kinetics: Relaxation Methods. in: Roberts, G. C. K. ed. Encyclopedia of Biophysics. Springer Berlin Heidelberg, pp. 1207-1212. Available at: http://dx.doi.org/10.1007/978-3-642-16712-6_62.
Geeves, M. and Lehrer, S. (2002). Cooperativity in the Ca2+ regulation of muscle contraction. in: Molecular Interactions of Actin. Springer-Verlag Berlin and Heidelberg GmbH & Co. K, pp. 111-132. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11892276.
Conference or workshop item
Boussouf, S. and Geeves, M. (2007). Tropomyosin and troponin cooperativity on the thin filament. in: 33rd Symposium on Regulatory Mechanisms of Striated Muscle Contraction. Berlin, Germany: Springer-Verlag Berlin, Heidelberger Platz 3, D-14197 Berlin, Germany, pp. 99-109. Available at: http://dx.doi.org/10.1007/978-4-431-38453-3_10.
The regulation of muscle contraction by the thin filament proteins tropomyosin (Tm) and troponin (Tn) has remained an area of interest since the proteins were first discovered 40 years ago.1,2 Although we have learnt a great deal about the proteins themselves and the mechanism by which they regulate muscle contraction some aspects of the mechanism remain to be adequately explained. Our interest is in the cooperativity of the calcium regulatory process and this remains poorly understood and several different models have been proposed. At it simplest the essence of the problem can be simply outlined. In skeletal muscle the binding of calcium to the two regulatory sites of TnC is required for activation of muscle contraction. Isolated TnC binds the calcium cooperatively, as might be expected for a two-calcium-ion switch, with a hill coefficient (h) of between 1 & 2.3,4 In contrast the calcium activation of isometric force in a skinned muscle fibre occurs with a much larger hill coefficient5,6 leading to the idea that cooperativity extends beyond the single actin7TmTn structural unit of the thin filament. Some models of muscle activation suggest the whole filament switches as a single unit while studies of the purified proteins in solution tend to indicate more limited cooperativity extending to only the nearest neighbour actin7TmTn units. In this paper the reasons why the nature of the cooperativity remains a problem will be explored together with an overview of what our recent studies of the proteins in solution have revealed about thin filament cooperativity.
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Molecular Motors
Myosins comprise one of the three major families of molecular motors which operate in cells. In addition to muscle contraction they are involved in a wide range cellular processes such as cell division, phagacytosis and vesicle transport. The understanding of the molecular events which make up the ATP driven cross bridge cycle (see fig) advances year by year yet there are continually new surprises in the variety and range of activities shown by members of the super family. We know know that myosins can move in opposite directions along actin filaments, can be processive and can act as cytoskeletal cross-linkers and strain sensors. Despite this variety in behaviour the same basic myosin motor domain brings about the different activities. Our work is directed towards understanding how the mechanochemistry of the myosin motor domain is tuned to produce these widely differing activities and how the motor activity is regulated.
Current Projects:
Mechanochemical coupling in the Myosin super family
The cross bridge cycle can be described as a series of coupled biochemical and mechanical events. The efficient conversion of biochemical energy requires a very precise temporal coupling between the biochemical and mechanical events and the structure of the myosin head is presumably designed to achieve this end. The cross bridge cycle for fast skeletal muscle is the most thoroughly studied system and the description of the cycle that follows refers to this actomyosin. The basic cycle is believed to be the same for all myosins but the relative rates of individual steps are altered by changes in the amino acid sequence of myosin to tune each myosin for its particular physiological role. Comparisons of the properties of different myosins together with mutagenesis of specific amino acid residues or of short sequences are the most active areas of current research.
Colour code for the Figure: Actin monomers - orange; Myosin head - green; Light chains - yellow; Myosin S2 & thick filament - black; ATP/ADP - blue/pink; Pi - pink/yellow.
The essential mechanochemical steps are shown lettered (a) to (e). ATP binding to either a rest length myosin head (c) or to a head bearing a load (b) results in a change in conformation of the myosin head causing a rapid almost irreversible dissociation of the myosin head from actin (d). Following detachment from actin the ATP is hydrolysed to ADP and Pi, both of which remain very tightly bound to the myosin head (e). The hydrolysis is relatively rapid (taking about 10 msec) and reversible (Keq = 10). The small value of the Keq indicates that the free energy of ATP hydrolysis is not released but remains within the structure of the M.ADP.Pi complex. The crystal structures of myosin heads with nucleotides or nucleotide analogues bound are believed to represent one or other of these two structures (d & e). This suggests that the hydrolysis is accompanied by a major conformational change which represents the reversal or a re-priming of the power stroke. Structures d & e are quite stable and ADP and Pi will remain bound to the myosin head until the myosin binds to an actin site. The affinity of M.ADP.Pi for actin is significantly higher than that of M.ATP and if an actin site is within reach of the myosin head it will bind rapidly and reversibly to the actin site and in doing so can explore several potential actin binding sites. Current opinion suggests that the majority (>80%) of myosin heads within an active isometric (not shortening) muscle are in equilibrium between states a d & e.
When the myosin head binds to actin the interaction with actin can promote a major change in conformation (the power stroke) which is accompanied by the dissociation of Pi. Crystal structures suggest that the power stroke consists of a reorientation of part of the myosin head distal to the actin-binding site and includes the converter domain and the light chain-binding region (LCBD). This results in the displacement of the distal tip of the LCBD by up to 10 nm. The structural changes in the actin-myosin interface that produces the power stroke remain undefined.
If the filaments carry an external load then the power stroke results in the distortion of an elastic element (b. The location and nature of the elastic element is unknown but may represent a distortion in the myosin head or of the LCBD. For simplicity the elastic element is drawn here as part of the connection between the myosin head and the thick filament. While the myosin head carries a load and is elastically distorted the dissociation of Pi is a reversible event and Pi can rebind to reverse the power stroke (and also back through intermediates e & d).
If the external load is small then the power stroke results in the relative sliding of the actin and myosin filaments by a distance of up to 10 nm. Following the sliding ADP is released very quickly (within 2 msec) to be replaced within a msec by ATP and the myosin head dissociates once more to complete the cycle. The final element of the mechanochemical coupling is believed to be a mechanism to limit the rate of release of ADP until the sliding motion is complete. Thus ADP release from b is much slower than from c. The mechanism of the strain limited ADP release is under current investigation but appears to be a key event which differs between myosins designed for efficient fast shortening vs. efficient load bearing. The structural changes observed on binding ADP to complexes of actin with smooth muscle myosin and brush border myosin I might reflect this strain sensing mechanism.
The mechanical model of the cycle has been updated more recently as more detailed structural information has become available.
Regulation of myosin motors
The regulation of the interaction between actin (A) and myosin heads (M ) in skeletal muscle is brought about by a Ca2+ induced change in state of the thin filament proteins tropomyosin (Tm) and troponin (Tn).1,2. The thin filament is a repeating structure made up of units of Actin7.TmTn. Skeletal tropomyosin is a linear coiled coil structure approximately 400 Å long which interacts with 7 actin subunits and the troponin regulatory complex. This structural repeat containing 7 actins per unit was used by Hill et. al.3 as the cooperative unit size in their model of thin filament cooperativity.
McKillop & Geeves4 more recently proposed that the regulation of TmTn containing thin filaments can be interpreted in terms of a rapid Ca2+ dependent equilibrium between 3 states of the thin filament, blocked, closed and open. A steric blocking model of thin filament regulation has been proposed (Figure) which shows the relationship between the three states of the thin filament and the two step binding of S1 to actin.5, 6, 7
1. Lehrer, S.S. (1994) J. Musc. Res. Cell Motil. 15, 232-236
2. Lehrer, S.S. and Geeves, M.A. (1998) J. Mol Biol. 277, 1081-1089
3. Hill, T.L., Eisenberg, E., and Greene, L. (1980) Proc. Natl. Acad. Sci USA 77, 3186-3190.
4. McKillop, D.F.A. and Geeves, M.A. (1991) Biochem. J. 279, 711-718.
5. Geeves, M.A., Goody, R.S. and Gutfreund H. (1984) J. Musc. Res. Cell. Motil. 5, 351-361.
6. Geeves, M.A. (1991) Biochem. J. 274, 1-14.
7. Geeves and Connibear (1995) Biophys. J. 68(4 Suppl), 194S-199S.
Inherited diseases of cardiac and skeletal muscle
There are a series of inherited human diseases of muscle caused by mutations in sarcomeric proteins such as the cardiomyopathies (hypertrophic and dilated cardiomyopathies) which can be life threatening or Freeman-Sheldon Syndrome which can cause developmental problems and major musculoskeletal deformities. The majority the cardiac mutations are found in myosin (~40% of known mutations) or myosin binding protein C (40%) with the rest in actin, tropomyosin, troponin, titin and other minor proteins. In most cases how the mutations cause the disease is not known.
Isolating or making the normal protein and the protein carrying the damaging mutation is one approach to understanding how the disease develops. Studying the protein in isolation can give some insight in to the problems caused by the mutation but studying the same protein in the context of its interacting partners provides much more detail whether this is in complex mixtures of proteins or in a contracting muscle.
We are studying the role of disease causing mutations in tropomyosin, troponin C, myosin and myosin binding protein C. Using the methods outlined elsewhere we can isolate the protein and reassemble the protein in complexes from filaments, myofibrils or in transgenic model systems like Drosophila flight muscle.
Development of novel fast reaction methods in skeletal and cardiac muscle contractiion
Pressure Jump
Rapid changes in pressure can be used to perturb the equilibrium position of many protein-protein or protein-ligand interactions on a time scale from 100 µs to several hundred seconds. Older equipment used volumes (1-2 mL) which are too extravagant for many biological materials. We have built a micro-volume pressure jump apparatus [Pearson et al. (2002) Biochem. J. 366(2): 643-651] based on the design of Clegg & Maxwell (1976) Rev. Sci. Inst. 47(11): 1383-93. The sample volume of the apparatus is 50 µL and it requires a minimum of 80 µL of sample for convenience of handling. The apparatus is designed with high pressure taps which allow simple rapid exchange of samples. Pressure is applied by means of a piezoelectric crystal stack separated from the sample by a polyimide membrane. Pressure jumps of up to +400 bar can be applied within 100 µs. The use of a piezoelectric stack allows the results of both pressure application and release to be repeatedly recorded and averaged, which allows small signal changes to be measured.
Fast Pressure Jump Equipment. A piston, driven by a piezoelectric stack, allows fast repetitive pressure jumps to be made on 50 µL of sample solution held within a sapphire ring. Typically, a photomultiplier measures fluorescence at 90° to the incident light (a simple rearrangement allows measurement of transmitted light instead).
Flash photolysis
Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However, these methods traditionally require relatively large amounts of protein (10-3g) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g., Dictyostelium). Single molecule and in vitro motility methods can be used on much smaller quantities but currently the range of measurements that can be made is limited. We have been developing methods which allow such fast transients to be studied on the much smaller quantities of protein (10-7, 10-6g) available from individual Drosophila muscles, single mammalian muscle fibers, human cardiac biopsies and non-muscle myosins.
Put the content of the callout in here. 
Flash Photolysis set-up: Simultaneous time resolved detection of transmission changes and either fluorescence or light scattering changes, for which different light sources and a different set of filters are used.
The quartz cuvette in the core of the system operates with a minimal sample volume of 10 µl. A Laser System with a wavelength of 347 nm provides the source of the flashlight causing the fast release of ATP from a caged compound into the solution. The optical bench is designed for simultaneous time resolved detection of the following transmission changes and of fluorescence (or light scattering) changes (90°) in the cuvette to monitor the reactions kinetics. For detection of fluorescence changes a Xe-Hg-lamp is used, while for detection of light scattering a halogen lamp is prefered. The detection electronics include two photomultipliers and a digital oscilloscope connected to a PC.
Acknowledgements:
Major Current Collaborations:
- Leslie Leinwand (University of Colorado, USA [http://biofrontiers.colorado.edu/about/directory/leinwand]),
- Mike Regnier (University of Washington, USA [http://www.bioeng.washington.edu/regnier/people/regnier.html]),
- Srboljub Mijailovich & Tom Irving (Illinois Institute of Technology, USA [https://science.iit.edu/people/faculty/thomas-irving]).
- Corrado Poggesi [http://www.unifi.it/p-doc2-2013-200009-P-3f2a3c2d332e2c-0.html] & Chira Tesi (University of Florence, Italy [http://www.unifi.it/p-doc2-2013-200009-T-3f2a3d2c342b2e-0.html])
Previous long-term collaborators:
- Drs Sam Lehrer & Lynne Coluccio, Boston Biomedical Research Institute
- Deitmar Manstein, Hannover Medical School [https://www.mh-hannover.de/bpc_prof_manstein.html]
- Rob Cross, University of Warwick [http://www2.warwick.ac.uk/fac/med/staff/cross/],
- David Smith Monash University Melbourne, and Sanford Bernstein, San Diego State University [http://bernstein-lab.sdsu.edu/index.html]
Year 1 BI300 Introduction to Biochemistry (Module convenor)
Year 2 BI503 Cell Biology
Final Year BI674 Molecular Machines in Biology (Module convenor)
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- Biophysical Society (USA)
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- American Society for Biochemistry and Molecular Biology. https://www.asbmb.org/
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