How does strain affect the contraction mechanism?
We are very interested in understanding the mechanism by which force affects the contractile machinery and the energy transduction process. This investigation requires functioning muscle fibres in which the arrangement of the proteins into filaments is preserved, the ability to measure fibre force, and to change the length of the fibre. In addition, we need to measure chemical changes: the rate at which Pi and ADP are released by the muscle cells. for this purpose, we use fluorescently-labelled proteins that bind Pi or ADP. These proteins can diffuse into the muscle cells, and their change in fluorescence measured through an epi-fluorescence microscope gives us a measure of the rate of ATP hydrolysis with millisecond time resolution. The structure of the fluorescently-labelled phosphate binding protein is shown below:
The protein has a fold into which Pi (shown in cyan) binds with high affinity. The Pi-cleft closes upon Pi-binding. The protein was genetically engineered to incorporate a cysteine near the Pi-cleft to allow labelling with a coumarin fluorophore (shown in red) : N-[2-(1-Maleimidyl)ethyl]-7- (diethylamino)coumarin-3-carboxamide (MDCC) which increases its fluorescence five-fold upon Pi binding (Brune et al., 1994). For ADP, we use a similar approach with a genetically-engineered, fluorescently-labelled nucleoside diphosphokinase (Brune et al., 2001). These techniques allow us to construct models of the actomyosin ATPase in muscle based on principles of enzyme kinetics. The data is used to calculate the rate constants for a reaction mechanism that can be represented as shown below:
where A represents Actin, M represents Myosin and where ki and ri are the forward and reverse rate constants for the ith steps (West et al., 2009).
