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 . AlF4- as FRET accepters of Mant fluorescence. The resultant estimated distances between loop RI and the active site differed for the Mant-S1 . TNP-ADP . BeFn and Mant-S1 . TNP-ADP . AlF4- 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.