Chemo-Mechanical Energy Transduction

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Morales 9603670 Technical This study examines the interface between actin and myosin S1 that lead to chemomechanical transduction of energy during muscle contraction. Thus site-directed mutagenesis is used to specify regions responsible for the hydrophobic and coulombic interactions, and to deduce the involvement of myosin ATPase. Specific questions are: 1 Why do the actin affinities for the "no ligand" and the MgATP" intermediates differ by a thousand fold? 2 Is it possible that only NMR methods can explain this conformational change, if this affinity difference is not due to charge, but to a myosin conformational change? and 3 How is the transition rate of the "MgADP" intermediate to the "no ligand" intermediate influenced by interaction with actin? Site-directed mutations and simulations with energy minimizations are used to elucidate the structural basis of the strong "no ligand" affinity. A structure of the "MgADP" intermediate with suitable attributes is simulated. The NMR chemical shift findings of Shriver/Sykes (At high T there is one, and at low T there are two coexisting conformers of myosin, detectable by 31-P and 19-F probes) will be followed to guide studies on a variety of intramolecular myosin crosslinks which trap these "MgADP" intermediary conformers, so that whether coexistence includes structures "missed" by current myosin crystallography can be determined. Furthermore, as an effort to understand actin activation of myosin ATPase, attempts are made to perturb structures that appear to affect the MgADP "leaving rate". The sites of interest are E99 and E100 for actin, K572, K574, D463 and Y135 for myosin, and a putative EDC crosslink site including D471; the activating effect of a bulky constituent (e.g. IATR) at C707 is energy-minimized. "Coexisting conformers such as the transitions between "ATP" and "ADP.Pi" are also examined. The significance of this study may lie in its molecularly defining the use of catalysis principles in energy-transduction in an important biological contratile system. Non-Technical The aim of this research is to understand the features and mechanisms that allow a biological entity to function as a machine - that takes in a fuel , transforms some of which into useful work, and generates others as waste , while the machine itself suffers no net change in this operational cycle. The paradigm being studied here is the biological machine used to contract muscle, particularly the involvement of the protein myosin which absorbs the fuel as well as ejects the waste. Static pictures in atomic detail of the structural states and flexibility of myosin are examined with physical methods such as nuclear magnetic resonance, comparing artificially altered and the natural wild versions. The effect of these alterations on operation is analyzed. Anchorage of the movement of the machine at each step by another protein, actin , is also studied. Although the central purpose of this research is to figure out how the muscle machine works, it is expected that the knowledge so gained will be used by researchers to understand, construct and repair biological machines in general, or to copy the extraordinary attributes of natural machines into man-made entities.