REGULATION OF CONTRACTION IN MUSCLE AND NONMUSCLE CELLS

研究的目的是了解细丝收缩调节的机理。肌凝蛋白充分激活细丝，Ca2+结合肌钙蛋白C在含有肌钙蛋白（Tn）的细丝，是合作的（变构）；原肌球蛋白（TM）是协同作用的促进剂。编码TM的N端和c端的交替表达外显子是TM通用和亚型特异性功能的主要决定因素，这与大量研究表明TM特异性TM功能的N端到c端相互作用，以及TM分子沿f -肌动蛋白的N端到c端相互作用对功能至关重要的结果一致。肌钙蛋白T结合到TM末端并改变TM的功能。TM和Tn基因突变可引起家族性肥厚性心肌病和线状性肌病。目前，关于TM和TnT的关键功能域的结构信息有限。利用圆二色光谱法和异核磁共振等生化方法，研究合成和重组N-和C-TM肽的结构、TnT的N端及其相互作用。关于TM和TnT的主要功能域结构的新知识将从拟议的工作中得到，这是揭示肌动蛋白丝和细胞骨架的基本和重要特性的关键一步：TM如何赋予细丝协同性以及信息如何沿着细丝协同交流。有四个具体目标。1. 确定由外显子1b编码的非肌肉α -原肌球蛋白N-末端的结构，并改进在肌原肌球蛋白中发现的外显子1a N-末端的结构。2. 测定原肌球蛋白c端结构。它形成了一个两股的线圈吗？3. 确定α / α原肌球蛋白N端和C端结构域之间的相互作用和重叠区域的结构。4. 确定人心脏TnT蛋白n端、原肌球蛋白结合调控域的结构，以及FHC突变对其结构和功能的影响。

The goal of the research is to understand the mechanism of thin filament regulation of contraction. The full activation of the thin filament by myosin, and Ca2+ binding to troponin C in thin filaments containing troponin (Tn), is cooperative (allosteric); tropomyosin (TM) is the agent for cooperativity. The alternatively expressed exons encoding the N- and C-terminal ends of TM are the major determinants of both universal and isoform-specific TM functions, consistent with extensive work showing that N- to C-terminal interactions of TM specific TM functions, consistent with extensive work showing that N-to C-terminal interactions of TM molecules along F-actin are essential for function. Troponin T binds to TM ends and modifies TM's function. Mutations in TM and Tn genes give rise to Familial Hypertrophic Cardiomyopathy and Nemaline Myopathy. At this time there is limited structural information about the critical functional domains of TM and TnT. Circular dichroism spectrometry and heteronuclear NMR, as well as other biochemical methods, will be used to investigate the structures of synthetic and recombinant N- and C-TM peptides, and the N-terminus of TnT, and the interactions between them. The new knowledge about the structure of the primary functional domains of TM and TnT that will come from the proposed work is a key step towards revealing a fundamental and significant property of the actin filament and cytoskeleton: how TM confers cooperativity to the thin filament and how information is communicated cooperatively along the thin filament. There are four specific aims. 1. To determine the structure of the non-muscle alpha-tropomyosin N- terminus encoded by exon 1b, and refinement of the structure of the exon 1a N-terminus, found in sarcomeric tropomyosins. 2. To determine the structure of the C-terminus of alpha/alpha- tropomyosin. Does it form a two-stranded coiled coil? 3. To determine the interaction and the structure of the overlap region between the N- and C- terminal domains of alpha/alpha-tropomyosin. 4. To determine the structure of the N-terminal, tropomyosin-binding, regulatory domain of human cardiac TnT, and the effect of FHC mutations on its structure and function.