This application seeks support for a research project in the investigation of the molecular mechanism of muscle contraction directed to an elucidation of the domain structure of the myosin head and its changes induced by nucleotide binding. This is a very important problem since nucleotide-induced interdomain interactions in the myosin head may plan an essential role in force generation in muscles. Earlier, the grant participants from Russian side applied the differential scanning calorimetry (DSC) method for a study of the myosin head structure, and revealed three separate structural domains in the myosin head. On the basis of results obtained and from the literature data, a domain model of the myosin head was suggested. It was shown also that the formation of the complex of the isolated myosin head (myosin subfragment 1, S1) with ADP and orthovanadate Vi (stable analogue of the intermediate complex S1-ADP-Pi, existing during the S1 steady-state ATPase reaction) results in a pronounced conformational change in the whole S1 molecule, reflected in a pronounced increase of S1 thermal stability and in a significant change of S1 domain structure. The DSC method, therefore, allows the S1 domain structure and the global conformational transitions induced by the formation of the complexes of the myosin head with nucleotide to be analyzed. On the other hand, the grant participants from American side have great experience in the field of investigation of the myosin head structure (preparation of isolated S1 fragments and light chains, different S1 modifications, usage of nucleotide analogues, etc). The collaboration with the Russian group will allow a more detailed analysis of the changes induced in the structure of S1 upon nucleotide binding since the DSC method will yield information about the global conformational transition due to nucleotide binding. The specific studies will include: (1) an investigation of changes induced by the reversible inactivation of S1 by near stoichiometric amounts of mercuric chloride; (2) an examination of the effect of SH-group modifications on the ability of S1 to undergo the global conformational transition due to the formation of S1-ADP-Vi complex; (3) use of different synthetic ADP analogues in order to investigate their ability to induce conformational changes in the S1 molecule in the complex with orthovanadate.
