PROTEINS & ARCHITECTURE OF AN ELASTIC SARCOMERE MATRIX

本建议的长期目标是了解结构和 横纹肌中肌节相关细胞骨架的生理作用 肌肉细胞 两个巨大的结构蛋白，肌联蛋白， 星云蛋白，表明每种蛋白质都构成了一组分子 纤维，在肌节中形成弹性基质。 肌节 基质可能发挥主要的生理作用，包括长 范围弹性，维持肌节稳定性和组装 在发育中的肌肉细胞中的新生肌节。 在拟议的研究中，我们解决了几个核心问题， 重要性，并以一种 综合实验方法： (a)肌联蛋白弹性的构象基础是什么？ 我们将测试 假设特异性和可逆构象转变之间 肌联蛋白多肽的折叠和未折叠状态是其作为 分子弹簧产生弹性。 分子结构及其 构象转换将在cDNA序列水平上确定， 氨基酸序列，蛋白质折叠，结构域组织，轮廓长度， 在各种实验条件下的灵活性和可扩展性。 (b)肌联蛋白和星云蛋白是否作为肌球蛋白的模板或支架， 肌动蛋白丝，分别？ 我们将寻找特定的丝间 蛋白质相互作用，并测试其对肌动球蛋白相互作用的影响。 此外，他们在集会中的潜在参与和长度 肌球蛋白和肌动蛋白丝的调节将在 自组装、成核组装和选择性重组 提取的肌节 (c)肌小节中肌联蛋白和星云蛋白是如何组织的？ 我们将评估A 四丝肌节模型的高分辨电镜观察 结合免疫标记。 此外，系统搜索 在Z-和M-线的结合蛋白将阐明其 锚定在肌节中。 (d)肌节基质如何对压力作出反应？ 我们 将评估 并完善我们的工作假设， 是它作为双射程分子弹簧的基础。 的电池 将使用针对非重复表位的单克隆抗体来跟踪 各种肌节中的动态易位和肌丝应变 长度 实验条件将被设计成便于 分子构象、肌节结构和 骨骼肌和心肌的肌肉力学。

The long range goal of this proposal is to understand the structural and physiological roles of sarcomere-associated cytoskeletons in the striated muscle cells. Recent studies of two giant structural proteins, titin, and nebulin, suggest that each protein constitutes a set of molecular filaments, forming an elastic matrix in the sarcomere. The sarcomere matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability and the assembly of nascent sarcomeres in developing muscle cells. In the proposed research, we address several questions of central importance and examine them systematically and critically with an integrated experimental approach: (a) What is the conformational basis of titin elasticity? We will test the hypothesis that specific and reversible conformational transition between folded and unfolded states of titin polypeptides underlies its capacity as a molecular spring to generate elasticity. The molecular structure and its conformational transition will be determined at the level of cDNA sequence, amino acid sequence, protein folding, domain organization, contour length, flexibility and extensibility under a variety of experimental conditions. (b) Do titin and nebulin serve as templates or scaffolds for myosin and actin filaments, respectively? We will search for specific interfilament protein interactions and test their effects on actomyosin interaction. Additionally, their potential involvement in the assembly and length regulation of myosin and actin filaments will be examined at the level of self-assembly, nucleated assembly and reconstitution of selectively extracted sarcomeres? (c) How are titin and nebulin organized in sarcomere? We will evaluate a four-filament sarcomere model by high resolution electron microscopy combined with immuno-labeling. Additionally, a systematic search for binding proteins at the Z-and M-lines will be made t elucidate their anchorage in the sarcomere. (d) How does the sarcomere matrix respond to stress? We will evaluate and refine our working hypothesis that the segmental extensibility of titin underlies its capacity as a dual-range molecular spring. A battery of monoclonal antibodies to non-repetitive epitopes will be used to track dynamic translocations and filament strain in sarcomeres of various lengths. Experimental conditions will be designed to facilitate correlation between molecular conformation, sarcomere architecture and muscle mechanics of skeletal and cardiac muscles.