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-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 Mg(2+)-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.
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249 Université de Montepellier I, France.
N
N
Bonafé
P
P
Chaussepied
J P
JP
Capony
J
J
Derancourt
R
R
Kassab
D000199:Actins / Q000737:chemistry*; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001386:Azides; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D010658:Phenylglyoxal / Q000031:analogs & derivatives; D010782:Photolysis; D011817:Rabbits
D000199:Actins; D001386:Azides; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010446:Peptide Fragments; C033025:actin subfragments; C032404:4-azidophenylglyoxal; D001120:Arginine; D017301:Ca(2+) Mg(2+)-ATPase; D010658:Phenylglyoxal