Conformational changes at the highly reactive cystein and lysine regions of skeletal muscle myosin induced by formation of transition state analogues.
Myosin forms stable ternary complexes with Mg2+-ADP and phosphate analogues of aluminum fluoride (AlF4-), beryllium fluoride (BeFn), and scandium fluoride (ScFn). These complexes are distinct from each other and may mimic different transient states in the ATPase cycle [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. Regions of skeletal muscle myosin containing the highly reactive residues Cys 707 (SH1), Cys 697 (SH2), and lysine 83 (RLR) dramatically alter their local conformation when myosin hydrolyzes ATP, and these changes may reflect formation of a series of transient intermediates during ATP hydrolysis. We used the fluorescent probes 4-fluoro-7-sulfamoylbezofurazan, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and trinitrobenzene-sulfonate, which bind to SH1, SH2, and RLR, respectively, to examine differences in local conformations within myosin.ADP.phosphate analogue (BeFn, Vi, AlF4-, and ScFn) complexes. It was observed that the ternary complexes had SH1 conformations similar to those seen on S-1 in the presence of ATP. In contrast, local conformations in the SH2 and RLR regions of S-1.ADP.BeFn were different from those in corresponding regions of S-1.ADP.AlF4- or ScFn. These results suggest that SH1 and SH2 move distinctly during ATP hydrolysis and that the local conformations of the SH2 and RLR regions more sensitively reflect different transient states.
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo, 192-8577, Japan. shinsaku@t.soka.ac.jp
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002645:Chickens; D003545:Cysteine / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000201:enzymology; D009218:Myosins / Q000737:chemistry*; D010851:Picrates / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents
D000817:Anilino Naphthalenesulfonates; D010851:Picrates; D013439:Sulfhydryl Reagents; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C005858:picric acid; D000251:Adenosine Triphosphatases; D009218:Myosins; D008239:Lysine; D003545:Cysteine