Nesmelov Yuri E, Agafonov Roman V, Burr Adam R, Weber Ralph T, Thomas David D
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN55455, USA.
Biophys J. 2008 Jul;95(1):247-56. doi: 10.1529/biophysj.107.124305. Epub 2008 Mar 13.
Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.
自旋标记和多频电子顺磁共振光谱被用于探测骨骼肌肌球蛋白在力产生区域的动态局部结构。兔骨骼肌肌球蛋白的亚片段1（S1）在C707（SH1）处用碘乙酰胺自旋标记进行标记。记录了无核苷酸状态以及存在ADP和核苷酸类似物时的X波段和W波段电子顺磁共振光谱。通过将电子顺磁共振光谱与模拟光谱拟合，根据肌球蛋白内自旋标记的旋转运动对其进行分析。考虑了两种模型：快速极限振荡（光谱仅取决于取向分布）和缓慢受限运动（光谱取决于旋转相关时间和取向分布）。对在两个微波频率（9.4 GHz和94 GHz）获得的光谱进行全局分析，为第二种模型提供了明确支持，并能够详细确定旋转运动的速率和幅度以及解析多个构象状态。无核苷酸生化状态可以用肌球蛋白的单一结构状态（M）很好地描述，自旋标记的运动非常受限且缓慢。肌球蛋白的ADP结合生化状态也揭示了单一结构状态（M*，先前已证明与动力冲程后ATP结合状态相同），自旋标记的运动受限较小且缓慢。相比之下，94 GHz时可用的额外分辨率表明，肌球蛋白的S1.ADP.V(i)结合生化状态（可能模拟S1.ADP.P(i)状态）的电子顺磁共振光谱清晰地解析为三个光谱成分（结构状态）。一种状态与ADP结合状态（M*）无法区分，其特征是适度受限和缓慢运动，摩尔分数为16%。其余84%（M**）包含另外两个成分，其特征是围绕自旋标记的x轴快速旋转。在分析了电子顺磁共振光谱、肌球蛋白ATP酶活性以及肌球蛋白II的可用结构信息后，我们得出结论，动力冲程后和动力冲程前的结构状态（M和M*）共存于S1.ADP.V(i)生化状态中。我们提出，动力冲程前状态M**的特征是两种结构状态，这可能反映了肌球蛋白转换器和N端结构域之间的灵活性。
