Analysis of conformational changes at the unique loop adjacent to the ATP binding site of smooth muscle myosin using a fluorescent probe

Recent crystallographic studies have shown that smooth muscle myosin has th ree highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyosteli um myosins, We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino ac ids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Bioche m. 124, 528-533], In the present study, in order to clarify the role of the se smooth muscle myosin loops in energy transduction, we specifically label ed the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methyla nthraniloyl)-8-azido-ADP (Mant-8-N-3-ADP), and then measured the fluorescen t polarization. When Mant-8-N-3-ADP was trapped by aluminium fluoride or va nadate into the ATPase site, Mant-8-N-3-ADP was covalently incorporated int o loop N (125-134), In contrast, Mant-8-N-3-ADP trapped by beryllium fluori de was covalently incorporated into both loop M (687-699) and loop N (125-1 34) at an almost equimolar ratio. Actin binding to smooth muscle myosin S1 (SMO-S1) labeled at only loop N (125-134) increased the polarization due to the viscosity of actin, In contrast, S1 labeled at both loops N and M show ed a much smaller increase in polarization, Our results indicate that the p robe at loop M (687-699) of smooth muscle myosin moved to a less hindered r egion, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skelet al muscle myosin, as previously demonstrated in our laboratory.