The long-term objective of the proposed work is to understand the molecular basis of the mechanism of muscle contraction. This will involve understanding how ATP hydrolysis is coupled to the large- scale macro-molecular changes responsible for force generation and how the changes are regulated. This work, which has a basic science objective, will generate knowledge which may have implications for cardiac and muscle-related disease. We proposed using neutron solution scattering and neutron helical diffraction to investigate specific structural questions crucial to the understanding of how muscle works. The experiments will utilize the selective deuteration of one, or sometimes two, of the members of the macromolecular complex under study. By varying buffer D2O content, contrast-matching of the buffer with protonated (H) or deuterated (D) protein will be achieved. This will allow examinations of unmatched protein (either H or D) in situ with little or no interference from the other (""""""""invisible"""""""") proteins. Deuterated proteins will be generated by cell biology and recombinant DNA techniques. We will investigate the following questions: (1) What is the in situ structure of regulatory light chains (RLC's) of myosin when the myosin heads are either free in solution or bound to actin with and without Ca2+? What is the separation of RLC's when heads ar bound to actin? (2) What is the in situ structure of troponin-C (TNC) with and without bound Ca2+ and what is the cross-helix radial separation of TNC with and without bound Ca2+? Is the TNC- TNC separation affected by myosin binding to the thin filament? (3) What are the structural changes, if any, that occur in the thin filament when myosin heads bind to actin in the absence of ATP? (4) What is the position and separation of RLC in relaxed, rigor, the putative weak-binding state and in an analog-induced state thought to resemble a force generating acto-myosin intermediate? (5) What is the change in position and intra-myosin separation of light chains when muscles are stretched in rigor? (6) What is the average radial position of the light chain-bearing portion of the myosin head in a muscle during isometric contraction of muscle fibers? Does the intensity of the 14.3 mn meridional reflection (arising from light chains alone) increase during contraction as it does in X-ray diffraction? (7) What are the phases of the equatorial neutron diffraction pattern from rigor vertebrate muscle? Can the low-resolution structure of rigor muscle fibers be determined from neutron diffraction? The answers to these questions will test hypotheses about how muscle contraction and regulation occur.
