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 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-N(3)-ADP), and then measured the fluorescent polarization. When Mant-8-N(3)-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-8-N(3)-ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N(3)-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.
Maruta
S
S
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan. shinsaku@t.soka.ac.jp
Saitoh
J
J
Asakura
T
T
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001608:Beryllium / Q000737:chemistry; D001665:Binding Sites; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D005459:Fluorides / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D015398:Signal Transduction; D062367:ortho-Aminobenzoates / Q000737:chemistry / Q000378:metabolism
D005456:Fluorescent Dyes; D062367:ortho-Aminobenzoates; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins; D001608:Beryllium; D005459:Fluorides