Objective
Direct observation of protein movements is difficult as few techniques have the required spatial and temporal resolution. Nevertheless the understanding of the molecular basis of muscle contraction requires such measurements. With X-ray diffraction the ordered packing of muscle proteins in skeletal muscle fibres results in spatial sampling of the diffracted X-rays, thus providing spatial resolution of a few nanometers. Using a bright X-ray source, time resolution on the time scale of the molecular events underlying muscle contraction can be achieved.
The aim of this research is to combine time-resolved X-ray diffraction with biophysical techniques to disrupt the equilibrium state of isolated muscle fibres, and to observe the time-course of induced structural and physiological changes, with the purpose of determining the structural basis of the cross-bridge power stroke, namely of the molecular events which underlie the conversion of chemical energy into work.
The research involves experiments with permeabilised muscle fibres isolated from vertebrate skeletal muscles. The experiment to determine the time-course of the change in the 143 angstoms meridional reflection in activated fibres following a change in muscle length will be carried out over a range of ATP concentrations. It will determine the role of ATP concentration on the rate and amplitude of the cross-bridge power stroke, and on the time-course of repriming. The effect of ATP concentration is expected to be particularly marked on the time-course of repriming as the completion of the ATP hydrolysis cycle is expected to take place during this phase. The ATP-dependence on the time-course of the return of the 143 angstoms to the isometric level will test the understanding of the structural changes underlying this repriming phase.
The physiological responses of permeabilised muscle fibres to rapid perturbations will be determined. Stiffness will be studied by the application of low- and high-frequency length perturbations (500 Hz and 3 kHz sine waves), and the results compared with stiffness determined from the application of single length steps. The change in stiffness following the photogeneration of ATP in rigor fibres will be investigated, both in the presence and absence of calcium. The effect of varying degrees of EDC cross-linking on stiffness will also be investigated with a view to quantifying the contribution of individual cross-bridges to overall fibre stiffness.
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NW7 1AA London
United Kingdom