PHOTOCHEMICAL CROSS-LINKING OF THE SKELETAL MYOSIN HEAD HEAVY-CHAIN TO ACTIN SUBDOMAIN-1 AT ARG95 AND ARG28

F-actin specifically substituted with the photocross-linker, p-azidoph enylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon c omplexation with myosin subfragment-1 (S1) and photolysis at 365 nm, i t was readily cross-linked to the Sl heavy chain with a yield of about 13-25%, generating four major actin-heavy-chain adducts with molecula r masses in the range 165 - 240 kDa. The elevated Mg2+-ATPase of the c ovalent complexes displayed a turnover rate of 33 +/- 8 s-1 which is s imilar to the values reported earlier for other acto-S1 conjugates. Th e cross-linking between various proteolytic Sl and actin derivatives, combined with the fluorescent and immunochemical detection of the phot ocross-linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50-kDa region, whereas that attached to Arg28 mediated the selective cross-linking of the COOH-ter minal 22-21-kDa fragments of the heavy chain, most probably by reactin g at or near the connector segment between the 50-kDa and 20-kDa fragm ents. The rapid photoactivation and cross-linking to Sl of the substit uted F-actin, which can be accomplished on a millisecond time scale, m ay serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain-1 of actin during the ATPase cycle.