LUMINESCENCE RESONANCE ENERGY TRANSFER ON ACTOMYOSIN

描述：本提案的重点是研究构象 使用我们最近介绍的肌动球蛋白复合物内的变化 发光共振能量转移（LRET）技术， 广泛使用的荧光共振能量转移。 初步结果 表明LRET能够精确测量相对较大距离 在肌动球蛋白，并表明LRET应普遍适用于 研究蛋白质在大复合物中的构象变化。 肌动球蛋白是一种 一个特别重要的分子马达， 变化导致肌肉收缩和各种亚细胞运动， 真核生物 在这里，研究人员建议使用LRET来确定两个 肌肉力学的基本参数。 首先，他们将测量S1肌球蛋白头和肌动蛋白丝之间的距离， 活跃的肌肉 目的是确定肌球蛋白头的分数， 在肌动球蛋白循环中与肌动蛋白结合并产生力。 这 参数已被其他工人广泛研究，但 确切的答案仍然难以捉摸。 其次，LRET将用于检测S1内的构象变化 头部，唱歌纯化蛋白质和活性纤维。 尽管人们普遍认为 将S1内涉及构象变化的肌肉力学视为 肌肉力学的“摆动-横桥”模型的组成部分， 很少直接测量这种构象变化。 具体来说，他们将寻找监管机构之间距离的变化， S1头部的轻链和核苷酸结合区。 如果目前的 模型是正确的，这个距离应该从大约51 A改变到 从作功冲程开始时的65 A到作功冲程结束时的65 A。 这两个测量将在等长条件下对肌肉进行， 但它们可以作为类似测量肌肉的模型， 长度变化。 LRET适用于这些测量，因为该技术可以直接 测量活动肌肉的构象变化， 感兴趣，并且相对非侵入性。 为了实现这些目标， 在某些情况下，LRET的技术进步。 将进行初步实验 镧系元素螯合物供体和光谱仪上的数据。 然而，首席研究员还将开发新的反应形式， 镧系元素螯合物，这将使位点特异性连接到S1和 其他蛋白质，测量大距离（大于100 A）的能力， 以及检测多种构象状态的能力。 他还将 将他们现有的基于比色皿的LRET光谱仪转换为 基于显微镜的光谱仪，用于测量单个肌原纤维， 纤维 这将包括增加一个CCD（电荷耦合探测器）， 测量供体和受体的发射光谱，这将增加 能量转移测量的灵敏度和准确度。

DESCRIPTION: The focus of this proposal is to study the conformational changes within the actomyosin complex using our recently introduced technique of luminescence resonance energy transfer (LRET), an extension of the widely used Fluorescence Resonance Energy Transfer. Preliminary results show that LRET is capable of accurately measuring relatively large distances in actomyosin, and indicates that LRET should be generally applicable to the study of protein conformational changes in large complexes. Actomyosin is a particularly important molecular motor in which protein conformational changes lead to muscle contraction and a variety of subcellular motion in eucaryotes. Here the investigators propose to use LRET to determine two essential parameters in muscle mechanics. First, they will measure distances the S1 myosin head and actin filaments in active muscle. The goal is to determine the fraction of myosin heads which are bound to actin, and generate force, during the actomyosin cycle. This parameter has been extensively investigated by other workers but a definitive answer remains elusive. Second, LRET will be used to detect conformational changes within the S1 head, sing purified proteins and active fibers. Despite the widely held view of muscle mechanics involving conformational changes within S1 as an integral part of the "swinging-crossbridge" model of muscle mechanics, there have been very few direct measurements of such conformational changes. Specifically, they will look for changes in distance between the regulatory light chain of the S1 head and the nucleotide binding region. If current models are correct, this distance should change from approximately 51A at the beginning of the power stroke, to 65A at the end of the power stroke. These two measurements will be made on muscle under isometric conditions, but they serve as models for similar measurements on muscle undergoing length changes. LRET is suited for these measurements because the technique can directly measure conformational changes in active muscle over the distances of interest, and is relatively non-invasive. To achieve these goals requires, in some cases technical advances in LRET. Initial experiments will be made on our current generation of lanthanide chelate donors and our spectrometer. However, the principal investigator will also develop new reactive forms of lanthanide chelates, which will enable site-specific attachment to S1 and other proteins, the ability to measure large distances (greater then 100A), and the ability to detect multiple conformational states. He will also convert their present cuvette-based LRET spectrometer into a microscope-based spectrometer for measurements on single myofibrils and fibers. This will include adding a CCD (charge-coupled-detector) for measurement of emission spectra of donor and acceptor, which will increase the sensitivity and accuracy of energy transfer measurements.