REGULATION OF CONTRACTION IN MUSCLE AND NONMUSCLE CELLS

本研究的目的是了解细丝收缩的调节机制。肌球蛋白对细丝的完全激活和钙离子与肌钙蛋白C的结合是协同作用的(变构)；原肌球蛋白(TM)是协同作用的介质。编码TM的N-末端和C-末端的交替表达的外显子是决定通用和同型特异性TM功能的主要因素，这与大量研究表明TM特异的TM功能的N-端到C端的相互作用是一致的，也与大量的工作表明TM分子沿着F-肌动蛋白的N-端到C端的相互作用是必不可少的。肌钙蛋白T与TM末端结合并修饰TM的功能。TM和TN基因突变导致家族性肥厚型心肌病和线虫性肌病。目前，关于TM和TNT的关键功能结构域的结构信息有限。利用圆二色谱和异核核磁共振等生物化学方法研究合成和重组N-TM和C-TM多肽的结构以及TNT的N-末端，以及它们之间的相互作用。从拟议的工作中获得的关于TM和TNT主要功能结构域的新知识是揭示肌动蛋白细丝和细胞骨架的基本和重要属性的关键一步：TM如何赋予细丝协作性以及信息如何沿着细丝协同传递。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，并对肌节原肌球蛋白中发现的外显子1a N-末端的结构进行精制。2.确定α/α-原肌球蛋白C-末端的结构。它是否形成双链卷曲？3.确定α/α-原肌球蛋白N-末端和C-末端结构域之间的相互作用和重叠区的结构。4.确定人心肌TnT的N端、原肌球蛋白结合、调节结构域的结构，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.