The project is aimed at furthering an understanding of the mechanism of myosin-linked regulation of contraction at the molecular level. By using combined spectroscopic and kinetic approaches it should be possible to identify both structural and dynamic aspects of the regulatory mechanism. The spectroscopic approach involves fluorescence energy transfer. This technique requires regulatory light-chains that are specifically modified with fluorescence probes and utilizes the known ability of such modifed light-chains to fully restore complete function to desensitized scallop myosin preparations. Using appropriate energy transfer couples, one on each myosin head, for each of a series of foreign regulatory light-chains labelled at different locations in their primary structure, it is hoped to establish the orientation of regulatory light-chains on the myosin head. Moreover, for hybrid myosin molecules using light-chains exhibiting substantial energy transfer it should be possible to examine potential changes in myosin head-head interactions under conditions that simulate rigor (no MgATP), rest (MgATP, no calcium) and the active state (MgATP, calcium). Any movement of one head relative to the other will manifest itself as a change in the degree of energy transfer. This approach will be complemented by the use of lanthanide ion probes. These studies will be conducted alongside binding and kinetic experiments designed to pinpoint the exact step of the actomyosin ATPase cycle that is blocked during myosin-linked regulation, when the muscle is at rest. Binding between actin and calcium-sensitive scallop myosin subfragments, as a function of actin concentration (MgATP (plus/minus) calcium), will be conducted in the airfuge alongside ATPase measurements made under identical conditions, after the fashion of Chalovich and Eisenberg (13). This approach will also be used to assess the function of scallop tropomyosin. The projects dscribed here complement a number of existing biochemical and structural approaches to thick filament regulation. A detailed understanding of such regulation is of fundamental importance.