Transient detection of spin-labeled myosin subfragment 1 conformational states during ATP hydrolysis
| Autor: | David D. Thomas, Howard D. White, E. M. Ostap |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Time Factors
Myosin light-chain kinase Protein Conformation Stereochemistry Population macromolecular substances Biochemistry Cyclic N-Oxides Myosin head Adenosine Triphosphate ATP hydrolysis Myosin medicine education Actin education.field_of_study Photolysis Meromyosin Chemistry Electron Spin Resonance Spectroscopy Myosin Subfragments Hydrogen-Ion Concentration Adenosine Diphosphate Kinetics Spectrometry Fluorescence Biophysics Spin Labels medicine.symptom Muscle contraction |
| Zdroj: | Biochemistry. 32:6712-6720 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00077a026 |
| Popis: | 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-S 1). Changes in the internal rotational dynamics of IASL-S 1 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 FM ATP) in the presence of 100 FM 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. The molecular mechanism of muscle contraction involves the direct interaction of myosin and actin, coupled to the hydrolysis of adenosine 5'-triphosphate (ATP). According to the rotating cross-bridge model, the pivoting motion of the myosin head attached to actin produces strain, which is then relieved by the sliding motion of the myosin and actin filaments (Reedyet al., 1965;Huxley, 1969;Huxley & Simmons, 1971). The general hypothesis is that changes in the nucleotide at the myosin active site induce changes in the conformation of the myosin head, which change the interaction between myosin and actin in a way that results in directional sliding of the filaments (Taylor, 1979; Eisenberg & Hill, 1985; Hibberd & |
| Databáze: | OpenAIRE |
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