title: High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label.
abstract: 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 α-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° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° 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.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455;
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Binder
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455;
authors.forename: Sinziana
authors.initials: S
authors.lastname: Cornea
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455;
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Thompson
authors.affiliation: Department of Chemistry and Geology, Minnesota State University, Mankato, MN 56001.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Moen
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455; ddt@umn.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013113:Spin Labels
chemical_list: D000199:Actins; D013113:Spin Labels; D009218:Myosins
keywords: BSL; actomyosin; electron paramagnetic resonance; muscle