Conformational Changes in the Unique Loops Bordering the ATP Binding Cleft of Skeletal Muscle Myosin Mediate Energy Transduction

Abstract

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-N₃-ADP and Mant-2-N₃-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_6V 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•Fn and TNP-ADP•AIF^-₄ as FRET acceptors of Mant fluorescence. The resultant estimated distances between loop M and the active site differed for the Mant-Sl•TNP-ADP•BeFn and Mant-S1•TNP-ADP•AIF^-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.