The contraction of muscle is known to occur by the interaction of the separate protein components, myosin and actin, of the two partially interdigitating myofilaments that comprise the contractile apparatus. Although it is thought that the force of contraction results from a change in the structure of the myosin molecule while it is hydrolyzing MgATP and interacting with actin, the nature of the structural changes is poorly defined. This proposal aims to examine the role of the myosin molecule and in particular of its two classes of light chains, the alkali light chains and the DTNB light chains, in the contractile function of fast-twitch skeletal myosin. The possibility will be examined that these two light chains are interacting within a myosin head and that changes in this interaction caused by the binding of Ca++ to the DTNB light chain and/or its phosphorylation results in alterations to both the SF2/SF1 junction and the actin binding domain of the heavy chain. This will be studied in two ways. First, the stability of the alkali light chain interaction in myosin and actomyosin will be examined in preparations devoid of, or containing, the DTNB light chains and, in the latter case, the effects of Ca++ binding and/or phosphorylation will be studied. Second, the spatial relationship of these two light chains in myosin will be examined by a detailed crosslinking approach and by energy transfer procedures. Evidence will also be sought for changes in this spatial relationship produced by actin binding, and by phosphorylation of, and Ca++ binding to the bound DTNB light chains. These studies should provide valuable information about the role that these light chains play in the molecular mechanism of contraction.
