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Research has been conducted on the photoaffinity spin-labeled non-nucleoaside ATP analogue. Results indicate that it is a substrate for skeletal myosin subfragment 1 and can be photoincorporated at the subfragment 1 active site.

Raman difference spectroscopic methods are used to derive the Raman spectra of the non-bridging, short, equatorial V--O bonds in the myosin S1-MgADP.Vi complex and model compounds in solution. Spectral changes observed were explained using previous experimental data and vibrational results from ab initio calculations. The structural results observed in the study and their ramifications on the reaction mechanism of myosin-catalyzed adenosine triphosphatase hydrolysis are discussed.

The mechanism of the vanadate-promoted photocleavage reaction at the active site of skeletal muscle myosin subfragment 1 is further studied. The technique involved the isolation of peptides from both the COOH and NH2 termini of the photocleaved site. Characterization of these peptides indicated that the COOH-terminal peptide was amidated and the NH2-terminal peptide was blocked byh an oxalyl group. A mechanism in which O2 adds to a free radical on the alpha-carbon of Ser-180 with a subsequent Criegee type rearrangement is suggested as the explanation for both the kinetics and products of the photocleavage.

Photoreactive analogues of ATP such as the arylazido-ADP analogue NANDP, have been used to identify amino acid residues and specific regions of the myosin heavy chain interacting with ATP. 2-azidoadenosine diphosphate (2-N3ADP) was used to label the purine binding site of ATP on skeletal myosin. There were eight photolabeled peptides which were then subjected to sequence and radiolabeling analysis. Results showed that Trp-130 was labeled by 2-N3ADP just like NANDP. The azido groups of the photolabeling compounds were also found to be superimposed if they bind to the active site in an extended conformation similar to the anti conformation of ATP.

The purine binding sites of filamentous scallop myosin were determined by photoaffinity labeling using the photoreactive ADP analog, 2-(4-azido-2- nitrophenyl)amino-ethyl diphosphate as the purine binding site label. The purine binding sites were localized to the amino acid residues Cys-198 and Arg-128. Decreasing the Ca2+ concentration resulted in preferential photolabeling at Arg-128. Desensitization of the scallop myosin did not alter the photolabeling pattern when photolyzed at different Ca2+ concentrations. These results indicate that Arg-128 and Cys-198 are purine binding sites which undergo conformational changes in response to Ca2+.

The phosphoryl-binding site of gizzard myosin in the 6S extended and 10S folded conformations was examined using the vanadate photomodification reaction. Photomodified 6S and 10S myosin were reduced to determine the site of photomodification and the amino acid contents were analyzed. Ser-179 was the only residue photomodified in both forms of the heavy chain of myosin that suggested similarity in the active site for both conformations. Results of the amino acid sequence showed the gamma-phosphate-binding site for ATP contained Ser-179 and proved the role of this region in phosphate-binding of ATP.