INTRINSIC PROBES OF SMOOTH MUSCLE MYOSIN DYNAMICS

描述（由申请方提供）：本提案的最终目标是使用内源性色氨酸荧光检查平滑肌肌球蛋白内特定位置的构象变化。平滑肌肌球蛋白构建体将被遗传工程化以在所需的感兴趣位点含有单个色氨酸或一对色氨酸，这将提供独特的内在荧光信号，报告响应于核苷酸结合、ATP水解、肌动蛋白结合和杠杆臂运动的局部构象和结构变化。这些实验是对该领域正在进行的结构研究的补充，使我们能够明确地测试在平滑肌肌球蛋白收缩周期的关键步骤中关于结构域运动和结构重排的预测。因此，我们将能够将肌球蛋白的结构变化与功能后果相关联，这与某些心血管疾病直接相关。例如，FHC（家族性肥厚型心肌病）是一种遗传性的，通常是致命的疾病，由肌球蛋白内关键结构的点突变引起，肌球蛋白作为分子马达的正常功能至关重要。我们将研究肌球蛋白的结构变化，这些结构变化直接受到FHC突变的影响。这将有助于更好地了解疾病，从而也有助于更好地选择治疗方案。因此，这项提议提供了一个独特的机会，可以批判性地测试有关肌肉收缩的分子机制的基本问题，这些问题以前无法通过其他光谱探针研究获得，并且结果将对严重疾病状态（如FHC）产生重要影响。

DESCRIPTION (provided by applicant): The ultimate goal of this proposal is to examine conformational changes at specific locations within smooth muscle myosin using intrinsic tryptophan fluorescence. Smooth muscle myosin constructs will be genetically engineered to contain either a single tryptophan or a pair of tryptophans at the desired site of interest, which will provide a unique intrinsic fluorescence signal reporting local conformational and structural changes in response to nucleotide binding, ATP hydrolysis, actin-binding, and lever arm movement. These experiments are complementary to the ongoing structural studies in the field, allowing us to explicitly test predictions about domain movements and structural rearrangements during critical steps in the contractile cycle of smooth muscle myosin. Thus we will be able to correlate structural changes in myosin with functional consequences, which relates directly to certain cardiovascular disease. For example, FHC (familial hypertrophic cardiomyopathy) is an inherited, often lethal disease caused by point mutations at key structures within myosin critical to its proper functioning as a molecular motor. We will be examining structural changes in myosin in regions of the molecule directly impacted by mutations that underlie FHC. This will lead to a better understanding of the disease, and thus to better treatment options as well. Therefore, this proposal offers a unique opportunity to critically test fundamental questions about the molecular mechanism of muscle contraction that have not been previously accessible by other spectroscopic probe studies, and the results will have important implications for serious disease states such as FHC.