Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.
To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5'-yl imidodiphosphate and ADP resulted in a significant decrease in the fluorescence intensity of approximately 40 and 30%, respectively. When ATP was added to MIANS-S-1, the fluorescence intensity decreased rapidly by approximately 40%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity increased to approximately 70% of the original value. These results together with model experiments with MIANS-N-acetylcysteine indicate that in the presence of ATP, the MIANS fluorophore attached to SH2 is located in a less hydrophobic environment than is the fluorophore in the absence of ligand and that the hydrolysis of ATP enhances hydrophobicity around the fluorophore. Acrylamide fluorescence quenching studies of MIANS-S-1 confirmed these results, indicating that addition of ATP and ADP to MIANS-S-1 results in an increase in the Stern-Volmer quenching constant of the fluorophore by factors of approximately 3 and 2.5, respectively. The present observations suggest that binding of ATP causes a movement of SH2 toward the protein surface, whereas it goes back into the protein interior after ATP hydrolysis. The results also confirmed previous observations by a chemical cross-linking approach (Hiratsuka, T. (1987) Biochemistry 26, 3168-3173).
Hiratsuka
T
T
Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D006868:Hydrolysis; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D006898:Hydroxylamines; D013439:Sulfhydryl Reagents; D019811:Hydroxylamine; D000244:Adenosine Diphosphate; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D000255:Adenosine Triphosphate; D009218:Myosins; D003545:Cysteine