Photochemical cross‐linking of the skeletal myosin head heavy chain to actin subdomain‐1 at Arg95 and Arg28

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Abstract
F-actin specifically substituted with the photocross-linker, p-azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment-1 (S1) and photolysis at 365 nm, it was readily cross-linked to the S1 heavy chain with a yield of about 13–25%, generating four major actin–heavy-chain adducts with molecular masses in the range 165–240 kDa. The elevated Mg2+-ATPase of the covalent complexes displayed a turnover rate of 33±8 s−1 which is similar to the values reported earlier for other acto-S1 conjugates. The cross-linking between various proteolytic S1 and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross-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-terminal 22–21-kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50-kDa and 20-kDa fragments. The rapid photoactivation and cross-linking to S1 of the substituted F-actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain-1 of actin during the ATPase cycle.