Molecular dynamics studies of muscle proteins

描述（由申请人提供）：确定肌肉蛋白产生力和运动的原子水平机制仍然是生理学的基本问题之一。最近对运动蛋白肌球蛋白不同构象的 X 射线结构的测定，为其功能机制提供了新的假设。然而，定义核苷酸、ATP 与肌球蛋白相互作用以产生力和运动的统一框架仍未确定。核苷酸 (ATP) 类似物可以扰乱底物与蛋白质的相互作用，并将扰动与收缩活性的调节相关联。这可以作为核苷酸水解驱动肌球蛋白构象变化从而导致运动的机制的探针。我们将使用分子动力学 (MD) 模拟来研究 ATP 和核苷酸类似物与肌球蛋白 X 射线结构的相互作用，作为核苷酸水解驱动肌球蛋白构象变化产生力和运动的机制的探针。我们的基本工作假设是，X 射线结构的定量分析可以深入了解其他方法无法识别的功能。活性位点处的核苷酸类似物的模拟将扰乱通常与 ATP 结合相关的核苷酸-蛋白质模式，并允许就蛋白质和底物的相互作用绘制进一步的结构-功能相关性。主要目标是将模拟分析与现有实验数据联系起来，并确定对我们的建模研究产生的假设的进一步实验挑战。额外的 MD 模拟将研究肌球蛋白二聚结构域和其他 α 螺旋卷曲线圈结构的热力学稳定性。二聚化结构域的稳定性以及对肌球蛋白头-头相互作用和功能调节的影响仍未解决，一些模型表明卷曲线圈的熔化对于功能至关重要。将采用定量MD模拟来分析不同肌球蛋白亚型的卷曲螺旋结构域的相对稳定性及其与功能的关系。除了肌球蛋白 II 之外，我们还将研究肌球蛋白 V 和肌球蛋白 VI。在后一种亚型中，不稳定的卷曲大肠杆菌结构域的存在被认为对于功能至关重要。该协议将扩展到研究卷曲螺旋调节蛋白原肌球蛋白的突变，该蛋白与家族性肥厚型心肌病（FHC）相关。对肌肉蛋白功能的原子水平理解有望为 FHC 和其他肌肉疾病带来更合理的治疗方法。对肌球蛋白 V 和肌球蛋白 VI 功能的进一步了解有望改善人类格里斯利综合征和基于肌球蛋白 VI 的感音神经性听力损失的治疗。

DESCRIPTION (provided by applicant): Determining the atomic level mechanism by which muscle proteins generate force and motion remains one of the fundamental questions in physiology. The recent determinations of the x-ray structures of different conformations of the motor protein, myosin, have provided new hypotheses as to its mechanism of function. However, a unifying framework defining the interaction of the nucleotide, ATP, with myosin to generate force and motion remains undetermined. Nucleotide (ATP) analogs allow one to perturb the interaction of the substrate with the protein, and correlate the perturbations with modulation of contractile activity. This can serve as a probe of the mechanism by which nucleotide hydrolysis drives the conformational changes in myosin that result in motility. We will use molecular dynamics (MD) simulations to investigate the interaction of ATP and nucleotide analogs with myosin x-ray structures as a probe of the mechanism by which nucleotide hydrolysis drives the conformational changes in myosin that generate force and motion. Our fundamental working hypothesis is that quantitative analyses of the x-ray structures can yield insights into function that other approaches have failed to discern. The simulation of nucleotide analogs at the active site will perturb the nucleotide-protein patterns normally associated with ATP binding, and allow further structure- function correlations to be drawn regarding the interaction of protein and substrate. A major goal will be to relate the simulation analyses to existing experimental data, and to identify further experimental challenges to the hypotheses generated by our modeling studies. Additional MD simulations will investigate the thermodynamic stability of the myosin dimerization domain and other alpha-helical coiled coil structures. The stability of the dimerization domain and the implications for myosin head-head interactions and the regulation of function remain unresolved, with some models suggesting a melting of the coiled coil as crucial to function. Quantitative MD simulations will be employed to analyze the relative stabilities of the coiled-coil domains of different myosin isoforms and their relationship to function. In addition to myosin II, we will investigate myosin V and myosin VI. In the latter isoform, the presence of an unstable coiled-coli domain has been hypothesized to be crucial for function. The protocols will be extended to study mutations in the coiled-coil regulatory protein, tropomyosin, which are associated with familial hypertrophic cardiomyopathy (FHC). An atomic level understanding of muscle protein function can be expected to lead to more rational therapies for FHC and other muscle diseases. Improved understanding of the function of myosin V and myosin VI can be anticipated to improve therapies for Griscelli syndrome and myosin Vl-based sensorineural hearing loss in humans.