Regulation of Contraction in Muscle and Nonmuscle Cells

描述(申请人提供)：许多细胞活动所需的基于肌动蛋白的收缩和运动功能由原肌球蛋白(TM)和肌球蛋白共同调节。原肌球蛋白(TM)是一种沿着肌动蛋白细丝结合的卷曲蛋白质。总体目标是了解肌动蛋白功能的潜在调控机制。其目的是确定TM和肌球蛋白的卷曲结构域的结构和动力学，这两个结构域对功能是重要的。在前一次授予期间的工作导致了这样的假设，即灵活性和与规范螺旋螺旋结构的偏离是机械作用中结合部位和区域的重要特征。主要的方法是多维核磁共振和圆二色谱。原肌球蛋白和TM模型多肽将作为理解螺旋卷曲类蛋白质结合相互作用的范例。概述了测定TM的重要功能结构域、肌钙蛋白T(TNT)的TM结合结构域的复合体以及肌球蛋白的机械力化学功能区域在溶液中的构象、稳定性、原子分辨结构和动力学的实验。 目的1.用核磁共振技术测定横纹肌TM的C-端到N-端“重叠”区域以及与TNT的TM结合域的三元复合体的结构和动力学。 目的2.利用核磁共振技术确定短的非肌肉TM亚型的N-端-C-端重叠复合体的结构，并与横纹肌重叠复合体进行比较。 目的3.研究肌动蛋白与肌动蛋白结合和肌动球蛋白功能调节所必需的TM内部区域的构象、结构和相互作用的序列依赖性。 目的4.将螺旋线圈的构象和稳定性的分析扩展到三种肌球蛋白头尾连接处附近的肌球蛋白杆区；评估了这三类肌球蛋白在机械性能上的专门化取决于螺旋线圈的差异的假说。

DESCRIPTION (provided by applicant): Actin-based contractile and motile function required for many cellular activities is cooperatively regulated by tropomyosin (TM), a coiled-coil protein that binds along the actin filament, and myosin. The overall goal is to understand the mechanism underlying regulation of actin function. The aims are to determine the structures and dynamics of coiled-coil domains of TM and myosin that are important for function. Work during the previous granting period led to the hypothesis that flexibility and deviations from canonical coiled-coil structure are important features of binding sites and regions involved in mechanical action. The primary methods will be multidimensional nuclear magnetic resonance and circular dichroism spectroscopy. Tropomyosin and TM model peptides will serve as paradigms for understanding binding interactions of the coiled-coil class of proteins. Experiments are outlined to determine the conformation, stability, and atomic resolution structures and dynamics in solution of important functional domains of TM, complexes with the TM binding domains of troponin T (TnT), and a region of myosin proposed to be important for mechanochemical function. Aim 1. To determine using NMR the structures and dynamics of the C-terminal to N-terminal "overlap" regions of striated muscle TM and the ternary complex with the TM-binding domain of TnT. Aim 2. To determine the structure of the N-terminal to C-terminal overlap complex of short, non-muscle TM isoforms using NMR and compare it with the striated muscle overlap complex. Aim 3. To study the sequence dependence of the conformation, structure and interactions of an internal region of TM important for actin binding and the regulation of the actomyosin function. Aim 4. To extend the analysis of coiled coil conformation and stability to the myosin rod region next to the head-tail junction in three classes of myosin; evaluation of the hypothesis that specializations in mechanical performance among these classes depend on variations in the coiled coil.