Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase.
The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues.
Hiratsuka
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Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014364:Tryptophan / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D013439:Sulfhydryl Reagents; D000244:Adenosine Diphosphate; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; D009218:Myosins; D003545:Cysteine