Professor Michael Geeves

Professor of Physical Biochemistry

About

Mike Geeves joined the School of Biosciences in January 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. He was Head of School between 2006 - 2010.
Mike is a member of the Mechanobiology group also known as MaDCaP 
ORCID ID: 0000-0002-9364-8898

Research interests

Molecular motors particularly the motors of the myosin family including the myosin IIs responsible for muscle contraction.
Regulation of muscle contraction via calcium.
Inherited diseases of skeletal and cardiac muscle.
Novel fast reaction methods.

Supervision

MSc-R project available for 2019/20

Evolution of the muscle sarcomere. A bioinformatics approach to the interaction between myosin and myosin binding protein-C 
(joint supervision with Dr Mark Wass)
Following on from a study of how muscle-type myosins have adapted, over evolutionary timescales, for different types of muscle contraction, we will explore the co-evolution of myosin and the myosin binding proteins C. MyBP-C is well known to carry mutations linked to inherited heart disease.
Additional research costs: £1200

Protein based temperature sensors for use inside cells and organelles (project jointly supervised with Dr Dan Mulvihill)
We have a created a series of prototype protein based sensors to allow variation and changes in temperature to be monitored within living cells.  Additional research costs: £1200

This project will;

  1. modify the protein to optimise the temperature profile for different cell types. 
  2. express the protein in a variety of prokaryote and eukaryote cells and organisms. 
  3. use signal peptides to target the sensor to specific cellular organelles. 

Live Cell Imaging under Pressure 
(project jointly supervised with Dr Dan Mulvihill)
We have a built a novel live cell imaging system that allows protein dynamics to be followed at 100 atmospheres, a pressure which specifically and reversibly disrupts protein dynamics. 

This project will use this system to follow actin and microtubule dynamics in a variety of cell types to study how perturbations of cytoskeleton dynamics affect growth and organisation of cells and development.
Additional research costs: £1200  

Publications

Showing 50 of 140 total publications in the Kent Academic Repository. View all publications.

Article

  • Hellerschmied, D., Lehner, A., Franicevic, N., Arnese, R., Johnson, C., Vogel, A., Meinhart, A., Kurzbauer, R., Deszcz, L., Gazda, L., Geeves, M. and Clausen, T. (2019). Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin. Nature Communications [Online] 10:4781. Available at: https://doi.org/10.1038/s41467-019-12667-8.
    Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.
  • Vera, C., Johnson, C., Walklate, J., Adhikari, A., Svicevic, M., Mijailovich, S., Combs, A., Langer, S., Ruppel, K., Spudich, J., Geeves, M. and Leinwand, L. (2019). Myosin Motor Domains Carrying Mutations Implicated in Early or Late Onset Hypertrophic Cardiomyopathy Have Similar Properties. Journal of Biological Chemistry [Online]. Available at: https://doi.org/10.1074/jbc.RA119.010563.
    Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, since HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (DR; the fraction of myosin in strongly attached force-holding states) and the measured ATPases all changed in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.
  • Baker, K., Gyamfi, I., Mashanov, G., Molloy, J., Geeves, M. and Mulvihill, D. (2019). TORC2-Gad8 dependent myosin phosphorylation modulates regulation by calcium. eLife [Online] 8. Available at: https://dx.doi.org/10.7554/eLife.51150.
    Cells respond to changes in their environment through signalling networks that modulate cytoskeleton and membrane organisation to coordinate cell cycle progression, polarised cell growth and multicellular development. Here, we define a novel regulatory mechanism by which the motor activity and function of the fission yeast type 1 myosin, Myo1, is modulated by TORC2 signalling dependent phosphorylation. Phosphorylation of the conserved serine at position 742 within the neck region changes both the conformation of the neck region and the interactions between Myo1 and its associating calmodulin light chains. S742 phosphorylation thereby couples calcium and TOR signalling networks in the modulation of myosin-1 dynamics to co-ordinate actin polymerisation and membrane reorganisation at sites of endocytosis and polarised cell growth in response to environmental and cell cycle cues.
  • Geeves, M., Lehrer, S. and Lehman, W. (2019). The Mechanism of Thin Filament Regulation: Models in Conflict?. The Journal of General Physiology [Online] 151. Available at: https://dx.doi.org/10.1085/jgp.201912446.
    In a recent article in this journal, Heeley and colleagues (Heeley, White, and Taylor 2019 J Gen Physiol 151, 628-634) reopened the debate about 2 vs 3 state models of thin filament regulation. The authors review their work, which measures the rate constant of Pi release from myosin.ADP.Pi activated by actin or thin filaments under a variety of conditions. They conclude that their data can be described by a 2-state model and raise doubts about the generally accepted 3-state model as originally formulated by McKillop and Geeves (Biophysical Journal 65: 693–701, 1993). However, in the following article, we follow Plato’s dictum that “twice and thrice over, as they say, good it is to repeat and review what is good”. We have therefore reviewed the evidence for the 3- and 2-state models and present our view that the evidence is overwhelmingly in favor of three structural states of the thin filament, which regulate access of myosin to its binding sites on actin and, hence, muscle contractility.
  • Johnson, C., Walklate, J., Svicevic, M., Mijailovich, S., Vere, C., Karabina, A., Leinwand, L. and Geeves, M. (2019). The ATPase cycle of human muscle myosin II Isoforms: adaptation of a single mechanochemical cycle for different physiological roles. Journal of Biological Chemistry [Online]. Available at: https://doi.org/10.1074/jbc.RA119.009825.
    Striated muscle myosins are encoded by a large gene family in all mammals, including human. These isoforms define several of the key characteristics of the different striated muscle fiber types including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin.myosin cross-bridge cycle in solution. Here we present data on an eighth isoform the perinatal, which has not previously been charaterized. The perinatal is distinct from the embryonic isoforms appearing to have features in common with the adult fast muscle isoform, including weak affinity of ADP for A.M and fast ADP release. We go on to use a recently developed modeling approach MUSICO to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction including duty ratio, shortening velocity, ATP economy and the load dependence of these parameters. The work shows that the parameters of the cross bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.
  • Walklate, J., Ujfalusi, Z., Behrens, V., King, E. and Geeves, M. (2019). A micro-volume adaptation of a stopped-flow system; use with μg quantities of muscle proteins. Analytical Biochemistry [Online]. Available at: https://doi.org/10.1016/j.ab.2019.06.009.
    Stopped-flow spectroscopy is a powerful method for measuring very fast biological and chemical
    reactions. The technique however is often limited by the volumes of reactants needed to load the
    system. Here we present a simple adaptation of commercial stopped-flow system that reduces the
    volume needed by a factor of 4 to ≈120 μL. After evaluation the volume requirements of the system
    we show that many standard myosin based assays can be performed using <100 μg of myosin. This
    adaptation both reduces the volume and therefore mass of protein required and also produces data
    of similar quality to that produced using the standard set up. The 100 μg of myosin required for
    these assays is less than that which can be isolated from 100 mg of muscle tissue. With this reduced
    quantity of myosin, assays using biopsy samples become possible. This will allow assays to be used
    to assist diagnoses, to examine the effects of post translational modifications on muscle proteins
    and to test potential therapeutic drugs using patient derived samples.
  • Dürst, C., Wiegert, J., Helassa, N., Kerruth, S., Coates, C., Schulze, C., Geeves, M., Török, K. and Oertner, T. (2019). High-speed imaging of glutamate release with genetically encoded sensors. Nature Protocols [Online] 14:1401-1424. Available at: https://doi.org/10.1038/s41596-019-0143-9.
    The strength of an excitatory synapse depends on its ability to release glutamate and on the density of
    postsynaptic receptors. Genetically-encoded glutamate indicators (GEGIs) allow eavesdropping on
    synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in
    detail. Based on the sensor iGluSnFR, we recently developed accelerated versions that allow
    investigating synaptic release during 100 Hz trains. Here we describe the detailed procedures for design
    and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic
    hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser scanning
    microscopy. As the released glutamate spreads from a point source - the fusing vesicle - it is possible to
    localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By
    using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak of fast
    iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.
  • Mijailovich, S., Stojanovic, B., Nedic, D., Svicevic, M., Geeves, M., Irving, T. and Granzier, H. (2019). Nebulin and Titin Modulate Cross-bridge Cycling and Length-dependent Calcium Sensitivity. The Journal of General Physiology [Online]. Available at: https://doi.org/10.1085/jgp.201812165.
    Various mutations in the structural proteins nebulin and titin that are present in human disease are known
    to affect the contractility of striated muscle. Loss of nebulin is associated with reduced actin filament
    length and impairment of myosin binding to actin, whereas titin is thought to regulate muscle passive
    elasticity and is likely involved in length-dependent activation. Here, we sought to assess the
    modulation of muscle function by these sarcomeric proteins by using the computational platform
    MUSICO (MUscle SImulation COde) to quantitatively separate the effects of structural changes, kinetics
    of crossbridge cycling, and calcium sensitivity of the thin filaments. The simulations show that variation
    in thin filament length cannot by itself account for experimental observations of the contractility in
    nebulin-deficient muscle, but instead must be accompanied by a decreased myosin binding rate.
    Additionally, in order to match the observed calcium sensitivity, the rate of TnI detachment from actin
    needed to be increased. Simulations for cardiac muscle provided quantitative estimates of the effects of
    different titin-based passive elasticities on muscle force and activation in response to changes in
    sarcomere length and inter-filament lattice spacing. Predicted force-pCa relations showed a decrease in
    both active tension and sensitivity to calcium with a decrease in passive tension and sarcomere length. We
    conclude that this behavior is caused by partial redistribution of the muscle load between active muscle
    force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, which
    together modulate the release of TnI from actin. These data help advance understanding of how nebulin
    and titin mutations affect muscle function.
  • Sparrow, A., Sievert, K., Patel, S., Chang, Y., Broyles, C., Brook, F., Watkins, H., Geeves, M., Redwood, C., Robinson, P. and Daniels, M. (2019). Measurement of Myofilament-Localised Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations. Circulation Research [Online] 124:1228-1239. Available at: https://doi.org/10.1161/CIRCRESAHA.118.314600.
    Rationale: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation, or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here we develop and characterise genetically encoded Ca2+ indicators restricted to the myofilament to directly visualise Ca2 changes in the sarcomere.

    Objective: To produce and validate myofilament restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil and levosimendan) or following co-transduction of two established hypertrophic cardiomyopathy (HCM) disease causing mutants (cTnT R92Q and cTnI R145G) that alter myofilament Ca2+ handling.

    Methods and Results: When expressed in adult ventricular cardiomyocytes RGECO-TnT/TnI sensors localise correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localised Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Co-adenoviral transduction of RGECO-TnT/TnI with HCM causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the two mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+.

    Conclusions: RGECO-TnT/TnI are functionally equivalent. They visualise Ca2+ within the myofilament and reveal unrecognised aspects of small molecule and disease associated mutations in living cells.
  • Mitrou, G., Sakkas, G., Poulianiti, K., Karioti, A., Tepetes, K., Christodoulidis, G., Giakas, G., Stefanidis, I., Geeves, M., Koutedakis, Y. and Karatzaferi, C. (2019). Evidence of functional deficits at the single muscle fiber level in experimentally-induced renal insufficiency. Journal of Biomechanics [Online] 82:259-265. Available at: https://doi.org/10.1016/j.jbiomech.2018.10.035.
    Chronic kidney disease patients present with metabolic and functional muscle
    abnormalities, called uremic myopathy, whose mechanisms have not yet been fully
    elucidated. We investigated whether chronic renal insufficiency (CRI) affects skeletal
    muscle contractile properties at the cellular level. CRI was induced surgically in New
    Zealand rabbits (UREM), with sham-operation for controls (CON), and samples were
    collected at 3 months post-surgery, following euthanasia. All protocols had University
    Ethics approval following national and European guidelines. Sample treatments and
    evaluations were blinded. Maximal isometric force was assessed in 382 permeabilized
    psoas fibers (CON, n=142, UREM, n=240) initially at pH7, 10oC (‘standard’
    conditions), in subsets of fibers in acidic conditions (pH6.2, 10oC) but also at near
    physiological temperature (pH7, 30oC and pH6.2, 30oC). CRI resulted in significant
    smaller average CSA (~11%) for UREM muscle fibers (vs CON, P<0.01). At
    standard conditions, UREM fibers produced lower absolute and specific forces (i.e.
    normalized force per fiber CSA) (vs CON, P<0.01); force increased in 30oC for both
    groups (P<0.01), but the disparity between UREM and CON remained significant.
    Acidosis significantly reduced force (vs pH7, 10oC P<0.01), similarly in both groups
    (in UREM by -48% and in CON by -43%, P>0.05). For the first time, we give
    evidence that CRI can induce significant impairments in single psoas muscle fibers
    force generation, only partially explained by fiber atrophy, thus affecting muscle
    mechanics at the cellular level.
  • Behrens, V., Walter, W., Peters, C., Wang, T., Brenner, B., Geeves, M., Scholz, T. and Steffen, W. (2019). Mg2+ -free ATP regulates the processivity of native cytoplasmic dynein. FEBS Letters [Online] 593:296-307. Available at: https://doi.org/10.1002/1873-3468.13319.
    Cytoplasmic dynein, a microtubule?based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 ?m. We also identified the ratio of Mg2+?free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.
  • Brooker, H., Gyamfi, I., Wieckowska, A., Brooks, N., Mulvihill, D. and Geeves, M. (2018). A novel live cell imaging system reveals a reversible hydrostatic pressure impact on cell cycle progression. Journal of Cell Science [Online] 131:jcs212167. 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.
  • Helassa, N., Durst, C., Coates, C., Kerruth, S., Arif, U., Schulze, C., Wiegert, J., Geeves, M., Oertner, T. and Torok, K. (2018). Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses. Proceedings of the National Academy of Sciences [Online]. Available at: https://doi.org/10.1073/pnas.1720648115.
    Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ?10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness but increased Kd for glutamate (137 ?M and 600 ?M, respectively). Compared with iGluSnFR, iGluu has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.
  • Geeves, M., Leinwand, L., Spudich, J., Ruppel, K., Kawana, M., Choe Yu, E., Svicevic, M., Mijailovich, S., Vera, C. and Ujfalusi, Z. (2018). Dilated cardiomyopathy myosin mutants have reduced force-generating capacity. Journal of Biological Chemistry [Online] 293:9017-9029. Available at: http://dx.doi.org/10.1074/jbc.RA118.001938.
    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 ?-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.
  • Johnson, C., Brooker, H., Gyamfi, I., O’Brien, J., Ashley, B., Brazier, J., Dean, A., Embling, J., Grimsey, E., Tomlinson, A., Wilson, E., Geeves, M. and Mulvihill, D. (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.
    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.
  • 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., Nedic, D., Svicevic, M., Stojanovic, B., Walklate, J., Ujfalusi, Z. and Geeves, M. (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.
    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 ?-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 ?-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 ?-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.
  • Mijailovich, S., Kayser-Herold, O., Stojanovic, B., Nedic, D., Irving, T. and Geeves, M. (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.
  • 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.
    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.
  • 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.
  • Walklate, J., Vera, C., Bloemink, M., Geeves, M. and Leinwand, L. (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/? 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.
  • 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.
  • Bloemink, M., Melkani, G., Bernstein, S. and Geeves, M. (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.
  • 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.
  • Lehman, W., Medlock, G., Li, X., Suphamungmee, W., Tu, A., Schmidtmann, A., Ujfalusi, Z., Fischer, S., Moore, J., Geeves, M. and Regnier, M. (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.
  • 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.
  • Nag, S., Sommese, R., Ujfalusi, Z., Combs, A., Langer, S., Sutton, S., Leinwand, L., Geeves, M., Ruppel, K. and Spudich, J. (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 ?-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human ?-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 ?-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 ?-cardiac myosin contractility as a result of the R403Q mutation.
  • 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.
  • Bloemink, M., Deacon, J., Langer, S., Vera, C., Combs, A., Leinwand, L. and Geeves, M. (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 ?-helix, helix O, and to Switch-2 via the fifth strand of the central ?-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 ?-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.
  • Lawrence, A., Taylor, S., Scott, A., Rowe, M., Johnson, C., Rigby, S., Geeves, M., Pickersgill, R., Howard, M. and Warren, M. (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.
    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).
  • Janco, M., Suphamungmee, W., Li, X., Lehman, W., Lehrer, S. and Geeves, M. (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.
  • Bloemink, M., Deacon, J., Resnicow, D., Leinwand, L. and Geeves, M. (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.
  • Mijailovich, S., Kayser-Herold, O., Li, X., Griffiths, H. and Geeves, M. (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.
  • Deacon, J., Bloemink, M., Rezavandi, H., Geeves, M. and Leinwand, L. (2012). Erratum to: Identification of functional differences between recombinant human ? and ? 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 ? and ? myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (?:?) while failing human ventricles express no detectable ?-myosin. We report here fast-kinetic analysis of recombinant human ? and ? myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of ?-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, ?-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow ? isoform despite 91% sequence identity between the motor domains of ?- and ?-myosin. Among the features that differentiate ?- from ?-S1: the ATP hydrolysis step of ?-S1 is ~ten-fold faster than ?-S1, ?-S1 exhibits ~five-fold weaker actin affinity than ?-S1, and actin·?-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for ?-S1. Overall, the cycle times are ten-fold faster for ?-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.
  • Janco, M., Kalyva, A., Scellini, B., Piroddi, N., Tesi, C., Poggesi, C. and Geeves, M. (2012). ?-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.
    ?-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 (??*) or two (?*?*) 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 ??* and ?*?* 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 ??*), similar to those of the mutant homodimer (calcium sensitivity, D175N ??*), intermediate between the two (actin affinity, E180G ??*), or different from both (thermal stability, E180G ??*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.
  • Deery, E., Schroeder, S., Lawrence, A., Taylor, S., Seyedarabi, A., Waterman, J., Wilson, K., Brown, D., Geeves, M., Howard, M., Pickersgill, R. and Warren, M. (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.
  • 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.
  • Canepari, M., Maffei, M., Longa, E., Geeves, M. and Bottinelli, R. (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 ?M for the fast isoform and 18.6, 36.5 and 45.5 ?M 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 ?M for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 ?M 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.
  • Mijailovich, S., Li, X., Griffiths, R. and Geeves, M. (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.
  • Deacon, J., Bloemink, M., Rezavandi, H., Geeves, M. and Leinwand, L. (2012). Identification of functional differences between recombinant human ? and ? cardiac myosin motors. Cellular and Molecular Life Sciences [Online] 69:2261-2277. Available at: http://dx.doi.org/10.1007/s00018-012-0927-3.
    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 ?rst such analysis of any human muscle myosin
    motor and the ?rst of a-myosin from any species. Our
    ?ndings 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 *?ve-fold weaker actin af?nity 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 ?nding.

Book section

  • 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.
  • 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.
  • Geeves, M. and Pearson, D. (2013). Kinetics: Relaxation Methods. In: Roberts, G. C. ed. Encyclopedia of Biophysics. Springer Berlin Heidelberg, pp. 1207-1212. Available at: http://dx.doi.org/10.1007/978-3-642-16712-6_62.
  • 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-?-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.

Thesis

  • Walklate, J. (2016). Kinetic Characterisation of Disease Causing Mutations in the Embryonic and ß-Cardiac Myosin Motor Domain.
    Myosin myopathies are a growing area of research not only to understand the nature of the disease and how it can occur, but also to gain insight into how the myosin molecule works. Point mutations are a great way of examining how regions of myosin interact, however, given that there are over 800 amino acids in the motor domain alone, pinpointing key residues can be challenging. The missense mutations in the myosin molecule that lead to disease are ideal then to investigate residue changes that will have an effect on the function of the motor. The expression of recombinant skeletal myosin class II molecules has only recently become possible.
    Previous studies into the function of the embryonic myosin isoform have shown it to be a slow type myosin similar to the ?-cardiac isoform. Here stopped-flow kinetic analysis of recombinant embryonic myosin S1 showed it has a tight ADP affinity and slow ADP release, characteristic of the ?-cardiac myosin. Analysis of the three most common mutations in the embryonic myosin that cause Freeman-Sheldon syndrome (R672H, R672C, and T178I) showed a significantly reduced ATP hydrolysis, and ATPase Vmax and KM. Modelling of the cycle found that the mutations will be detached from actin for longer due to reduced ATP hydrolysis rate and a slower estimated phosphate release step.
    Another more common myopathy is hypertrophic cardiomyopathy (HCM) which can be cause by mutations in a multitude of sarcomeric proteins, most notably the ?-cardiac myosin. HCM is usually found in adolescents and young adults; however cases are beginning to emerge involving young children. Stopped-flow kinetic analysis of one of these mutations, H251N, shows more significant effects on the myosin function than 'adult' HCM mutations, including; a weaker ADP affinity, tighter ATP affinity, and slower detachment from actin rate constant. However the difference in severity is not apparently clear from the stopped-flow data alone.
    These results highlight new key areas on the myosin molecule that are essential for its correct function. The myosin motor is an intricate machine with multiple parts that need further investigation to truly understand its function and the impact of disease causing mutations
  • Coghlan, M. (2015). The Analysis of Myosin II Evolution in Mammals.
    Myosin-II is a family of myosin that contains fifteen different isoforms that vary in function, from maintaining a critical role in sarcomeric contractions to non-muscle movement. Myosin-IIs structure can be divided in to two key domains, the motor domain and the tail domain. It is the motor domain that contains the proteins catalytic activity. The observed phenomenon of an inverse relationship between organism size and heart rate tells us that heart rates do vary with size. The larger the animal mass, the slower the heart rate. This phenomenon allowed us to propose the idea that variations in the sequence of cardiac isoforms could be observed as mass of the species increases, due to the variance of heart rates. If so, any divergence seen in the sequence of the ?-cardiac isoform would be reflected as species mass increases should not be seen in other isoforms that do not rely on heart rate variability. Through analysing the sequences of twelve mammalian species of varying mass, a strong divergence relationship was observed in the ?-cardiac motor domain with a correlation coefficient of -0.945 that was no observed in other isoforms. Key results show that myosin sequence divergence does have a marked dependence on both evolutionary separation and size difference between species.

Forthcoming

  • Vera, C., Johnson, C., Walklate, J., Adhikari, A., Ujfalusi, Z., Svicevic, M., Mijailovich, S., Combs, A., Langer, S., Ruppel, K., Spudich, J., Leinwand, L. and Geeves, M. (2019). Pediatric and adult-onset HCM mutations in the myosin motor domain have similar properties. BioRXiv (pre-print server) [Online]. Available at: https://doi.org/10.1101/622738.
    Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder that typically involves left ventricular hypertrophy and abnormal cardiac contractility. Mutations in β-MyHC are a major cause of HCM and are typically characterized with cardiac hypercontractility, but the specific mechanistic changes to myosin function that lead to the disease remain incompletely understood. Predicting the severity of any single β-MyHC mutation is hindered by a lack of detailed evaluation at the molecular level. In addition, since the cardiomyopathy can take 20 - 40 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations which result in childhood cardiomyopathies may show a more severe indication of molecular changes in myosin and be therefore easier to identify. In this work, we performed steady-state and transient kinetics analysis of the myosin carrying one of eight miss sense mutations in the motor domain. Five of these have been identified in childhood cardiomyopathies. The derived parameters were used to model the ATP driven cross bridge. Contrary to our hypothesis, the results show no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted A·M·D occupancy for [A] = 3 Kapp along with the closely related Duty Ratio (DR) and the measured ATPases all change in parallel (in both sign and degree of change) compared to the WT values. Six of the eight HCM mutations are clearly distinct from a set of DCM mutations previously characterized.
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