REGULATION OF CONTRACTION IN MUSCLE AND NONMUSCLE CELLS

本研究的目的是了解细丝调节收缩的机制。肌球蛋白对细丝的完全激活，以及Ca 2+与含有肌钙蛋白（Tn）的细丝中的肌钙蛋白C结合，是协同的（变构的）;原肌球蛋白（TM）是协同性的试剂。交替表达的外显子编码的N-和C-末端TM的通用和异构体特异性TM功能的主要决定因素，与大量的工作表明，TM特异性TM功能的N-到C-末端的相互作用，与大量的工作表明，TM分子的N-到C-末端的相互作用沿着F-肌动蛋白是必不可少的功能。肌钙蛋白T与TM末端结合并改变TM的功能。TM和Tn基因突变引起家族性肥厚型心肌病和线状肌病。目前，关于TM和TnT关键功能域的结构信息有限。圆二色光谱和核磁共振，以及其他生物化学方法，将被用来研究合成和重组的N-和C-TM肽的结构，和TnT的N-末端，以及它们之间的相互作用。关于TM和TnT的主要功能结构域的结构的新知识，将来自拟议的工作是一个关键的一步，揭示了肌动蛋白丝和细胞骨架的一个基本的和重要的属性：TM如何赋予合作性的细丝和信息是如何合作地沟通沿着细丝。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，以及在肌节原肌球蛋白中发现的外显子1a N-末端结构的细化。2.确定α/α-原肌球蛋白C-末端的结构。它会形成一个双股卷曲螺旋吗？3.确定α/α-原肌球蛋白N-和C-末端结构域之间的相互作用和重叠区的结构。4.目的：研究人心肌肌钙蛋白T（TnT）N-末端原肌球蛋白结合调节区的结构，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.