MULTIFREQUENCY ESR SPECTRA OF SPIN-LABELED PROTEIN FROM MD SIMULATIONS

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 多频电子自旋共振 (ESR) 谱提供了有关自旋标记局部环境的丰富结构和动力学信息，并间接提供了有关它们所附着的蛋白质的信息。然而，将观察到的光谱特征与潜在的分子运动和相互作用明确联系起来是具有挑战性的。 为了对 ESR 光谱进行严格的解释，我们对完全溶剂化的 T4 溶菌酶进行了广泛的分子动力学 (MD) 模拟，在位置 72 和 131 处进行了自旋标记。这两个位点一直是大量实验研究的对象，并且已将其自身确立为 α 螺旋表面上典型的溶剂暴露位点。 为了延长模拟提供的时间窗口，根据轨迹构建了随机马尔可夫模型，反映了自旋标签侧链在旋转异构状态方面的动态。 在没有可调参数的情况下，在三个不同磁场强度下计算的位置 72 和 131 的多频 ESR 谱与实验结果在数量上一致。 在动力学过程中，自旋标记可以获得相当大量的构象，并显示出与螺旋上最近的邻居以外的蛋白质残基形成相互作用的显着倾向。 MD 模拟中出现的自旋标签的详细图像统一了不同的光谱和晶体学数据，并提供了对其分子起源的前所未有的洞察。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Multifrequency electron spin resonance (ESR) spectra provides a wealth of structural and dynamics information about the local environment of the spin label, and indirectly about the proteins to which they are attached. Unambiguously relating the features of the observed spectra to the underlying molecular motions and interactions is, however, challenging. To progress toward a rigorous interpretation of ESR spectra, we performed extensive molecular dynamics (MD) simulations of fully solvated T4 Lysozyme, labeled with spin labeled at positions 72 and 131. These two sites have been the object of numerous experimental studies, and have established themselves as prototypical solvent-exposed sites on the surface of alpha-helices. To extend the time window afforded by the simulations, stochastic Markov models, reflecting the dynamics of spin label side chains in terms of their rotameric states, are constructed from the trajectories. Without adjustable parameters, the multifrequency ESR spectra calculated at three different magnetic field strengths for positions 72 and 131 were found to be in quantitative agreement with experiment. During dynamics the spin label has access to a fairly large number of conformations and displays a significant propensity to form interactions with protein residues other than the nearest neighbors along the helix. The detailed picture of the spin label emerging from the MD simulations unifies the diverse spectroscopic and crystallographic data and provides unprecedented insight into their molecular origins.