Conformational changes in the 23-kilodalton NH2-terminal peptide segment of myosin ATPase associated with ATP hydrolysis.
A fluorophore, 9-anthroyl (AN) group, was covalently incorporated into the 23-kDa NH2-terminal peptide segment of myosin subfragment-1 (S-1) (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194). The fluorescent S-1 derivative (AN-S-1) was utilized to detect conformational changes in the 23-kDa segment associated with ATP hydrolysis of S-1. The extrinsic fluorescence of AN-S-1 was sensitive to binding of ligands. Upon addition of adenyl-5'-yl-imidodiphosphate (AMP-PNP), pyrophosphate (PPi), and ADP, fluorescence of AN-S-1 decreased by 10, 34, and 50%, respectively. Using this fluorescence decrease, the number of ligand binding sites (n) and the dissociation constant (KD) for the binary complexes of AN-S-1 were obtained: n = 0.96, KD = 15 microM for AMP-PNP; n = 1.0, KD = 5.0 microM for PPi; n = 0.99, KD = 27 microM for ADP. When ATP was added to AN-S-1, the fluorescence intensity decreased rapidly by about 30%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity decreased further to 50% of the original level. Model experiments with AN-N-acetylserine suggest that the fluorophore attached to S-1 is gradually exposed to more hydrophilic surface of protein with a progress of the ATPase reaction. The results indicate that conformational changes associated with ATP hydrolysis occur in the vicinity of the fluorophore attached to the 23-kDa NH2-terminal peptide segment of S-1.
Hiratsuka
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Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D000873:Anthracenes; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D013050:Spectrometry, Fluorescence
D000873:Anthracenes; D005456:Fluorescent Dyes; D008024:Ligands; D015879:Myosin Subfragments; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C066291:9-anthroylnitrile; D000255:Adenosine Triphosphate; D009218:Myosins