Conformational Transitions in Myosin Subfragment-1

Ushakov, Dmitriy S., Conformational transitions in myosin subfragment-1. Doctor of Philosophy (Biochemistry), June 2000, 72 pp., 7 tables, 19 illustrations, bibliography: 139 titles. The contraction of muscles is driven by ATP-dependent interaction of actin and myosin filaments. It has been recently shown that the regulatory domain (RD) of smooth muscle myosin, containing both the regulatory and essential light chains, exists in different orientations depending on the nucleotide bound to the myosin ATPase site. However, this could not be detected in skeletal muscle myosin, and therefore it is still not known whether it is the RD or the change in the myosin motor domain (MD) that is responsible for the force production. To investigate this, we used chemical cross-linking to analyze the binding of myosin subfragment-1 (S1) to F-actin in the presence of various adenine nucleotides. We found that ADP causes the reorientation of S1 with respect to F-actin, but only at physiological molar ratio of S1 to actin. The result can be simply explained by the two-state model of S1 binding to F-actin proposed earlier, in which S1 binds to one (state 1) or two (state 2) actin monomers, depending on the saturation of the filaments with S1. This suggests that the change in the orientation of RD could be a mere consequence of the conformational change in the MD. To investigate the changes in the RD further, we used a fluorescence anisotropy of an external fluorophore attached to a specific cysteine residue of the protein. To facilitate experiments, a tag of 6 histidines was genetically introduced at the C-terminus of LC1. The recombinant LC1 was labeled with rhodamine at the cysteine 178 near the C-terminus, and exchanged into free S1 or in muscle fibers. The fluorescence anisotropy showed that the LC1 becomes more immobilized in the presence of ATP compared to the rigor state. The fact that ATP increases immobilization of LC1 suggests that the conformational changes take place in the RD of S1 during the ATP hydrolysis. The ordering of the LC1 could be due to the ATP-induced closure of the cleft between a small β-sheet on LC1 (Cys178-Met145) and a flexible loop on the catalytic domain (Arg18-Arg24). From presented evidences, we conclude that the conformational transitions in both the MD and RD of S1 contribute to the power stroke.