Conformational changes in the unique loops bordering the ATP binding cleft of skeletal muscle myosin mediate energy transduction.
Myosin has three highly-conserved, unique loops [B (320-327), M (677-689), and N (127-136)] at the entrance of the ATP binding cleft, and we previously showed that the effects of actin are mediated by a conformational change in loop M [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, loops M and N were photolabeled respectively with fluorescent probes Mant-8-N(3)-ADP and Mant-2-N(3)-ADP in order to study conformational changes in the loops related to energy transduction. The effect of actin on the conformation of loop N was examined by analyzing fluorescence polarization and acrylamide quenching; the results were then compared with those previously reported for loop M. In contrast to loop M, the fluorescence polarization and the value of K(sv) of the Mant-groups crosslinked to loop N were slightly affected by actin binding. To study conformational changes in loops M and N during the ATPase cycle, FRET was analyzed using TNP-ADP.BeFn and TNP-ADP. AlF(4)(-) as FRET acceptors of Mant fluorescence. The resultant estimated distances between loop M and the active site differed for the Mant-S1.TNP-ADP.BeFn and Mant-S1.TNP-ADP.AlF(4)(-) complexes, whereas the distances between loop N and the active site differed slightly. These findings indicate that the conformation of loop M changes during the ATPase cycle, suggesting that Loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites. Loop N, by contrast, is not significantly flexible.
Maruta
S
S
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan. shinsaku@t.soka.ac.jp
Homma
K
K
D020106:Acrylamide / Q000737:chemistry; D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004735:Energy Transfer; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008958:Models, Molecular; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D062367:ortho-Aminobenzoates / Q000378:metabolism
D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D019920:Photoaffinity Labels; 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; D000251:Adenosine Triphosphatases