Muscle group - Myosin V project

Peter Knight, Matt Walker, Stan Burgess and John Trinick

For the past ~50 years the central problem of muscle contraction has been to understand the molecular mechanism of force production. In 1968 Hugh Huxley put forward the tilting cross-bridge hypothesis which proposes that force originates from a change in angle of the heads of the myosin molecules whilst attached to actin, fuelled by ATP hydrolysis. Still unproven, this hypothesis is widely believed, although now seems more likely that the major conformational change is within the myosin head rather than in the angle made with actin. The single most important reason why it has been difficult to test it is that, although the tightly bound states towards the of the 'power-stroke' are easy to study, the weaker states near the start of the stroke tend to dissociate at the low protein concentrations feasible in vitro.

We have been studying, by electron microscopy, the structures of a non-muscle isoform of myosin, myosin V, complexed with actin. Single myosin V molecules carry cargoes in a variety of cell types, including nerve, and have evolved to walk 'processively' without detachment. The weakly bound states of its power-stroke are as a consequence less easily dissociated. The heads of myosin V are also twice as long as the more commonly studied myosin II from muscle, making them easier to study by electron microscopy. Indeed, it was proposed that their large size would allow them to span the repeat of the actin filament helix and thereby walk with a straight rather than a helical trajectory. We obtained the first detailed micrographs of myosin V attached to actin by both heads, showing that the molecule can indeed span the actin helix and walk straight. Significantly, the leading head has a new attached conformation on actin. This is similar in shape to the crystal structure of myosin II heads in ADP-aluminium fluoride, which was thought to mimic the weak ADP.Pi state. Our data therefore provide strong support for the tilting bridge hypothesis. In order to improve the signal-to-noise in our data, the micrographs were subjected to a new variation of single particle image processing that treats windowed filament segments as the particles.

Figure: Single particle image processing of leading and trailing heads of myosin V attached to actin by both heads. The direction of walking is to the right. Note the bent pre-power shape of the lead heads. The variance image is shown below. Scale bar 36 nm.

 

 

Collaborators

Drs James Sellers, Fei Wang and John Hammer III (National Institutes of Health, Bethesda) and Dr Howard White (Eastern Virginia Medical School).

Publications

Walker, M. L., Burgess, S. A., Sellers, J. R., Wang, F., III, J. A. H., Trinick, J., and Knight, P. J. (2000). Two-headed Binding of a Processive Myosin to F-actin, Nature, 405, 804-807

Burgess, S. A., Knight, P. J., Walker, M. L., Schmitz, S., Sparrow, J. C., and Trinick, J. (2000). Real-space 3-D reconstruction of frozen-hydrated arthrin and actin filaments at 2 nm resolution, Biophys. J., 78, LA47

Funding:

We acknowledge the support of the BBSRC and NIH (USA) for this research.