文章基本信息

标题：High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label
本地全文：下载
作者：Benjamin P. Binder ; Sinziana Cornea ; Andrew R. Thompson 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：26
页码：7972-7977
DOI：10.1073/pnas.1500625112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe interaction between actin and myosin is responsible for driving a vast array of essential biological processes, including the production of force in contracting muscle. However, the structural behavior of the two proteins in complex is not well understood, because high-resolution atomic models are not yet available by traditional methods. We use a bifunctional spin label and site-directed electron paramagnetic resonance spectroscopy to determine orientations of individual -helices within the complex. We thus quantify for the first time, to our knowledge, structural changes within the motor domain of actin-bound myosin on nucleotide binding and dissociation. Our results provide valuable insight into the mechanism of muscle contraction while showcasing a method with wide applicability to other oriented biological systems. 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 -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.
关键词：muscle ; actomyosin ; electron paramagnetic resonance ; BSL