Analysis of the conformational change of myosin during ATP hydrolysis using fluorescence resonance energy transfer.
In order to elucidate the molecular basis of energy transduction by myosin as a molecular motor, a fluorescent ribose-modified ATP analog 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ATP (NBD-ATP), was utilized to study the conformational change of the myosin motor domain during ATP hydrolysis using the fluorescence resonance energy transfer (FRET) method. The FRET efficiency from the fluorescent probe, BD- or AD-labeled at the reactive cysteine residues, SH1 (Cys 707) or SH2 (Cys697), respectively, to the NBD fluorophore in the ATP binding site was measured for several transient intermediates in the ATPase cycle. The FRET efficiency was greater than that using NBD-ADP. The FRETs for the myosin.ADP.AlF4- and myosin.ADP.BeFn ternary complexes, which mimic the M*.ADP.P(i) state and M.ATP state in the ATPase cycle, respectively, were similar to that of NBD-ATP. This suggests that both the SH1 and SH2 regions change their localized conformations to move closer to the ATPase site in the M*.ATP state and M**.ADP.P(i) state than in the M*.ADP state. Furthermore, we measured energy transfer from BD in the essential light chain to NBD in the active site. Assuming the efficiency at different states, myosin adopts a conformation such that the light chain moves closer to the active site by approximately 9 A during the hydrolysis of ATP.
Mizukura
Yoshiaki
Y
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
Maruta
Shinsaku
S
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006868:Hydrolysis; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation
D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins