Quenching of fluorescent nucleotides bound to myosin: a probe of the active-site conformation.
The conformation of the active ATPase site of myosin subfragment 1 (S1) and actomyosin in myofibrils was probed by measuring the solvent accessibility of the bound ethenonucleotides epsilon ADP and epsilon ATP (during steady-state hydrolysis). Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent-phase quencher acrylamide, 25-400 mM. The fraction of the nucleotides that were specifically bound to the active site was determined following sedimentation in the presence and absence of 5 mM ADP. In agreement with previous investigations, both epsilon ATP and epsilon ADP were almost completely protected from the quencher when bound to the active site of myosin. The solvent accessibility of both epsilon ADP and epsilon ATP varied with both temperature and ionic strength. The nucleotides became more accessible at higher temperatures and higher ionic strength. At 1 M KCl the quenching curve was biphasic, indicating that the nucleotide pocket of myosin can exist in both a closed form that allows little quenching and a more open form that allows considerable quenching. However, the transition between forms was not strongly coupled to the state of the nucleotide, with a similar protection observed for both epsilon ADP and for epsilon ATP during steady-state cycling. epsilon ADP bound to acto-S1 or to actomyosin in myofibrils displayed the same degree of protection as seen with S1 alone. A similar result is obtained during steady-state hydrolysis. Thus nucleotides in the myosin pocket do not become more accessible to the solvent when myosin binds to actin in either rigor-ADP or active complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
Franks-Skiba
K
K
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Hwang
T
T
Cooke
R
R
D020106:Acrylamide; D000178:Acrylamides; D000205:Actomyosin / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D008956:Models, Chemical; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012997:Solvents
D000178:Acrylamides; D004985:Ethenoadenosine Triphosphate; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D012997:Solvents; D020106:Acrylamide; C015195:1,N(6)-ethenoadenosine diphosphate; D000244:Adenosine Diphosphate; D000205:Actomyosin; D009218:Myosins