INTRINSIC PROBES OF SMOOTH MUSCLE MYOSIN DYMAMICS

本建议的最终目的是使用固有色氨酸荧光检查平滑肌肌球蛋白特定位置的构象变化。平滑肌肌球蛋白构建体将通过基因工程在所需的感兴趣位点包含单个色氨酸，这将提供独特的固有荧光信号，报告响应于核苷酸结合、ATP水解和肌动蛋白结合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，使我们能够在平滑肌肌球蛋白收缩周期的关键步骤中明确测试有关区域运动和结构安排的预测。因此，我们将能够将肌球蛋白的结构变化与功能后果联系起来，这与某些心血管疾病直接相关。例如，FHC（家族性肥厚性心肌病）是一种遗传性的，通常是致命的疾病，由肌球蛋白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。我们将检查肌球蛋白分子区域的结构变化，这些区域直接受到FHC突变的影响。这将使人们更好地了解这种疾病，从而找到更好的治疗方案。因此，这一建议提供了一个独特的机会来严格测试肌肉收缩分子机制的基本问题，这些问题以前没有被其他光谱探针研究获得，并且结果将对严重疾病状态（如FHC）具有重要意义。

The ultimate goal of this proposal is to examine conformation changes at specific locations within smooth muscle myosin using intrinsic tryptophan fluorescence. Smooth muscle myosin constructs will be genetically engineered to contain a single tryptophan 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, and actin-binding. These experiments are complementary to the ongoing structural studies in the field, allowing us to explicitly test predictions about domain movements and structural arrangements during critical steps in the contractile cycle 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.