Muscles contract by the interdigitation of protein filaments within them. These filaments are made up of myosin and actin molecules. The head part of each myosin molecule is thought to interact with the actin filament in a cyclic manner driven by ATP hydrolysis, with each stroke of the cycle causing about 10nm of movement.

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The process of actomyosin sliding can be observed under the light microscope using a number of tricks to overcome the resolution barrier. Fluorescently-labelled actin filament sliding over a track of myosin molecules can be visualised using in vitro motility assays. The head of the myosin molecule  has been crystallised in several conformations which suggests that bending in the neck region, when the head is bound to actin, may be responsible for the power stroke.

We are using protein engineering (site-directed mutagenesis) to introduce fluorescent probes into a cellular myosin from Dictyostelium discoideum. These probes can detect conformational changes during single cycles of the myosin ATPase using fluorescence stopped-flow kinetic methods.

In vitro motility assay showing the sliding of actin filaments over a myosin surface initiated by flash photolysis of caged ATP.

Sliding of rhodamine labelled actin over myosin thick filaments in the presence of the Cy3-EDA-ATP. After the right-hand actin filament settles on a myosin filament, flash photolysis of caged ATP is used to initiate sliding and to displace the prebound Cy3.ADP.Pi from the myosin filament (Bagshaw and Conibear, 1999).