Transient detection of spin-labeled myosin subfragment 1 conformational states during ATP hydrolysis.
We have used time-resolved electron paramagnetic resonance spectroscopy and caged ATP to detect nucleotide-induced changes in the conformational state of spin-labeled myosin heads (IASL-S1). Changes in the internal rotational dynamics of IASL-S1 were monitored with millisecond time resolution during the pre-steady-state phase of ATP hydrolysis. The changes in the internal protein dynamics were rigorously correlated with specific biochemical kinetic transitions, allowing us to observe directly the dynamic sequence of structural changes in IASL-S1 during the binding and hydrolysis of ATP. When caged ATP was photolyzed (producing 500 microM ATP) in the presence of 100 microM IASL-S1, the EPR signal intensity rose transiently to the steady-state ATPase level, indicating increased rotational motion about the SH1 region of the myosin head. Kinetic and spectral analyses have resolved two phases of this transient, one representing the population of the M*.ATP state and the other representing the population of the M**.ADP.Pi state. We conclude that two motionally distinct states of the myosin head are present during ATP hydrolysis and that these states represent distinct conformational states that can be correlated with specific biochemical intermediates. Since specific labeling of myosin heads with IASL has been achieved in skinned muscle fibers, this study establishes the feasibility for the first direct detection and resolution of myosin's conformational transients during muscle contraction.
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism*; D010782:Photolysis; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013997:Time Factors
D003497:Cyclic N-Oxides; D015879:Myosin Subfragments; D013113:Spin Labels; C003028:N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; D000244:Adenosine Diphosphate; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate