The conformation of the active site of myosin probed using mant-nucleotides.
Changes in the conformation of the active site of myosin subfragment-1 (S1) may be linked to the production of force during the powerstroke. We probed the conformation of the nucleotide pocket by measuring the solvent accessibility of bound mant-nucleotides. Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent phase quencher acrylamide. The fluorescent mant moiety is attached to the ribose and is located near the outside of the pocket where it is likely to be sensitive to opening of the pocket. MantADP was highly protected from the quencher when bound to the active site of S1. A similar degree of protection was also observed for mantATP during steady-state hydrolysis by S1, and for mantADP bound to acto-S1 or to myosin in myofibrils. Assuming that S1-mantATP and actoS1-mantADP represent states at the beginning and the end of the powerstroke, respectively, we conclude that the myosin nucleotide pocket does not undergo a large conformational change during the powerstroke. However, the high degree of protection seen for mant-nucleotides is not easily explained by the open structure of the nucleotide pocket in the S1-nucleotide complex observed by x-ray diffraction.
Franks-Skiba
K
K
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Cooke
R
R
D020106:Acrylamide; D000178:Acrylamides; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D005456:Fluorescent Dyes; D006868:Hydrolysis; D066298:In Vitro Techniques; D008954:Models, Biological; D009119:Muscle Contraction / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates
D000178:Acrylamides; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D062367:ortho-Aminobenzoates; D020106:Acrylamide; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate