The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Analysis of Conformational Changes at the Unique Loop Adjacent to the ATP Binding Site of Smooth Muscle Myosin Using a Fluorescent Probe
Shinsaku MarutaJunya SaitohTsuyoshi Asakura
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2000 Volume 127 Issue 2 Pages 199-204


Recent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N3ADP), and then measured the fluorescent polarization. When Mant-8-N3-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-S-N3 ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N3-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) 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 showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skeletal muscle myosin, as previously demonstrated in our laboratory.

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