期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 112, 期 26, 页码 7972-7977出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1500625112
关键词
muscle; actomyosin; electron paramagnetic resonance; BSL
资金
- National Institutes of Health (NIH) [AR32961, AG26160]
- NIH [T32 AR007612]
- Minnesota State University
- Mankato Faculty Research Grant
Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known alpha-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1 degrees accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2 degrees accuracy. We used this approach to examine the crucial ADP release step in myosin's catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.
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