Strucutre of the UNC-45 Chaperone and its Interaction with Skeletal Muscle Myosin

描述(由申请人提供)：我们建议研究UNC-45分子伴侣的结构和作用机制，UNC-45分子伴侣是一种肌球蛋白附属的“ucs结构域”蛋白。分子伴侣通过帮助蛋白质折叠和抑制蛋白质变性和聚集，在肌肉发育和功能中发挥关键作用。我们已经证明，UNC-45对于骨骼肌肌球蛋白的积累和肌原纤维的组装是至关重要的，它与肌球蛋白相互作用以保护其免受热诱导的聚集。然而，UNC-45的结构、它与肌球蛋白的相互作用方式以及它在骨骼肌疾病中的作用在很大程度上还没有被探索。我们的目标是确定果蝇UNC-45的分子结构，并研究其与肌球蛋白的物理相互作用。我们将检验以下假设：1)ATP结合，增强UNC-45伴侣功能，导致蛋白质的结构元件发生构象变化；2)UNC-45的UCS域与肌球蛋白相互作用。为此，我们将通过结晶、X射线衍射和计算分析，在原子水平上研究脱氧核糖核酸键合态的UNC-45。这将是第一个高分辨率的ucs结构域蛋白，这是一类在真菌和哺乳动物中发现的肌球蛋白相关蛋白。我们还将通过负染、电子显微镜和单粒子图像分析将UNC-45与肌球蛋白S-1复合成像，然后将晶体结构对接到类平均投影中。这项研究计划将利用我们在收缩蛋白分析方面的专业知识和高素质合作者的能力。我们的研究将阐明UNC-45的结构，了解其与ATP的相互作用，并定义ATP诱导的构象变化。此外，我们的努力将深入了解UNC-45‘S与肌球蛋白底物的物理相互作用，这对肌原纤维组装和抗应力至关重要。与公共卫生相关。UNC-45伴侣对于肌球蛋白的功能完整性至关重要，肌球蛋白是肌肉的分子马达。我们将研究UNC-45分子伴侣的结构及其与其肌球蛋白靶标的相互作用。我们的努力将阐明UNC-45的作用机制，并对其在正常肌肉中的功能以及对其在肌肉应激中的作用提供深入的了解。

DESCRIPTION (provided by applicant): We propose to study the structure and mechanism of action of the UNC-45 molecular chaperone, a myosin- affiliated "UCS domain" protein. Molecular chaperones play key roles in muscle development and function by aiding protein folding and inhibiting protein denaturation and aggregation. We have demonstrated that UNC-45 is critical for skeletal muscle myosin accumulation and myofibril assembly and that it interacts with myosin to protect it from heat-induced aggregation. However, the structure of UNC-45, its mode of interaction with myosin and its roles in skeletal muscle disease are largely unexplored. Our goal is to define the molecular structure of Drosophila UNC-45 and to study its physical interaction with myosin. We will test the hypotheses that 1) ATP binding, which enhances UNC-45 chaperone function, causes structural elements of the protein to undergo conformational change and 2) the UCS domain of UNC-45 interacts with myosin. To this end, we will examine UNC-45 in the apo- and nucleotide-bound states at atomic-level resolution by crystallization, x-ray diffraction and computational analysis. This will be the first high-resolution structure of a UCS domain protein, a class of myosin-associated proteins found in fungi through mammals. We will also image UNC-45 complexed with myosin S-1 by negative staining, electron microscopy and single particle image analysis followed by docking the crystal structures into class averaged projections. This research program will take advantage of our expertise in contractile protein analysis and the capabilities of highly qualified collaborators. Our studies will elucidate the structure of UNC-45, provide an understanding of its interaction with ATP and define ATP- induced conformational changes. Further, our efforts will yield insight into UNC-45's physical interaction with the myosin substrate, which is critical to myofibril assembly and resistance to stress. PUBLIC HEALTH RELEVANCE. The UNC-45 chaperone is critical for the functional integrity of myosin, the molecular motor of muscle. We will study the structure of the UNC-45 chaperone and its interaction with its myosin target. Our efforts will elucidate the mechanism of action of UNC-45 and provide insight into how it functions in normal muscle as well as an understanding of its role during muscle stress.