Regulation of Contraction in Muscle and Nonmuscle Cells

[unreadable] 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. [unreadable] [unreadable] 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. [unreadable] 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. [unreadable] 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. [unreadable] 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. [unreadable] [unreadable]