A unique feature of striated muscle is its enormous diversity of fiber types. Much of the functional diversity is due to a range of myosin isoforms that help determine muscle speed. Sequence comparisons and functional studies at the molecular level have suggested specific structural domains of the myosin heavy chain modulate kinetic steps of the crossbridge cycle that presumably affect muscle speed. We will exploit unique features of the Drosophila system for investigating relationships between isoform differences in myosin head structure and muscle kinetics. A major advance will be the use of myofibrillar preparations that allow precise manipulation of sarcomere length and ion concentrations (calcium, MgATP, MgADP and phosphate), from which we can deduce kinetic constants of the acto-myosin cross-bridge cycle with high precision. The research addresses 3 basic questions: (i) what step(s) of the crossbridge cycle establishes the major kinetic differences between a very fast muscle and a very slow muscle? (ii) do fiber types with intermediate speeds have crossbridge rate constants between those of the extremes? and (iii) what steps of the myosin crossbridge cycle are affected by changes in specific variable region(s) between myosin isoforms? To answer these questions (framed as hypotheses), we will contrast kinetic schemes obtained from myofibrils of indirect flight muscle (IFM, an extremely fast muscle), jump muscle (a fast muscle), IFM myofibrils transgenically expressing myosin from embryonic body wall muscle (EMB, a very slow muscle), and IFM myofibrils expressing chimeras of IFM and EMB myosin. By correlating kinetic differences to specific structural regions (including the converter region), we will deduce molecular mechanisms based on the latest structural models of myosin. The major strengths of this investigation are the fully integrated approach (from single molecules to whole animal) made possible by employing Drosophila, and the use of myofibrils which bridges a gap between experiments with isolated myosin and skinned fiber experiments.
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