Professor Michael Geeves
Professor of Physical Biochemistry
School of Biosciences
- 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.back to top
Also view these in the Kent Academic Repository
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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.
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
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).
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.
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.
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)back to top
- Biochemical Society (UK)
- Biophysical Society (USA)
- Physiological Society (UK)
- American Chemical Society www.acs.org
- American Society for Biochemistry and Molecular Biology. https://www.asbmb.org/
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