PROTEIN EXCHANGE TO STUDY MUSCLE FUNCTION AND DISEASE

描述(摘自申请者的摘要)：肌肉调节 收缩很难研究，因为它涉及几个方面的变化 在一个高度有序的系统中的蛋白质。生化研究提供了 组件的详细信息，但会造成组织的损失。 纤维研究保留了这种结构，但几乎没有提供关于 组件的功能。通过在体内交换天然的调节蛋白 单纤维中含有蛋白质的探针，就有可能研究 光纤中单个组件的功能。打开荧光探头 在溶液研究中，肌钙蛋白和原肌球蛋白被证明是 对钙结合或活性肌动蛋白的部分敏感 配置。当放置在单纤维中时，荧光探针可以 用于在各种情况下监控调节装置的状态 机械扰动。这使得人们可以遵循个人的监管 蛋白质在纤维的组织结构中，并回答许多 与监管有关的问题。使用的交换方法是 与较旧的方法相比有所改进，并保留了 纤维没有变化。我们的计划是研究交流过程，以便 将其优化并扩展到横纹肌中的其他蛋白质。一个数字 的荧光探针将放置在肌钙蛋白的不同区域 络合物和纤维中发生的荧光变化将是相关的 与那些在受控条件下在溶液中发生的那些。此外 为了引入荧光探针，将采用交换过程 将其他特性良好的修饰或突变的肌钙蛋白引入 研究肌钙蛋白特定区域的功能。其意图是 是从检查监管突变的影响开始的 在肥厚型心肌病患者中发现的仪器。这项工作是 与Bernhard Brenner博士合作的一部分(机械 研究)、余力波博士(结构研究)和陈义德博士 (数学建模)。

DESCRIPTION (Adapted from applicant's abstract): Regulation of muscle contraction is difficult to study because it involves changes in several proteins that are in a highly organized system. Biochemical studies provide detailed information of the components but at the loss of the organization. Fiber studies preserve the structure but give little information about the function of the components. By exchanging native regulatory proteins in a single fiber with proteins containing a probe, it is possible to study the function of single components within a fiber. Fluorescent probes on troponin and tropomyosin have been shown, in solution studies, to be sensitive to calcium binding or to the fraction of actin in the active configuration. When placed into single fibers, the fluorescent probes can be used to monitor the state of the regulatory apparatus during various mechanical perturbations. This allows one to follow individual regulatory proteins in the organized structure of the fiber and to answer many questions related to regulation. The method of exchange that is used is an improvement over older methods and leaves the mechanical properties of the fibers unchanged. The plan is to study the exchange process so that it may be optimized and extended it to other proteins in striated muscle. A number of fluorescent probes will be placed on different regions of the troponin complex and the fluorescence changes that occur in fibers will be correlated with those occurring in solution under controlled conditions. In addition to the introduction of fluorescent probes, the exchange process will be used to introduce other well characterized modified or mutant troponins into fibers to study the function of particular regions of troponin. The intent is to begin by examining the effect of the mutations of the regulatory apparatus found in patients with hypertrophic cardiomyopathy. This work is part of a collaborative effort with Dr. Bernhard Brenner (mechanical studies), Dr. Leepo C. Yu (structural studies) and Dr. Yi-der Chen (mathematical modeling).