The aim of this interdisciplinary program is to determine the structure- function relationships and mechanisms of proteins important in normal cardiovascular function and subject to abnormalities that cause disease. Major components (Projects 1, 3 and 4) address the mechanism and structure of the small GTP-binding protein RhoA, its effector proteins and its mechanisms of regulation. RhoA regulates vascular smooth muscle contraction, cardiac hypertrophy and formation of stress fibers, and its downs stream effectors, Rho-kinase, was recently implicated in hypertension. The cellular mechanisms of RhoA and Rho-related proteins will be determine in Project 1, their atomic structures in Project 3, and their subcellular localization in Project 4, with proteins largely produced in Core B. The mechanisms of Ca/2+-desensitization protein, telokin, will be determined in Projects 1 and 3, and it sub-cellular localization in Project 4. The molecular structure of myosin, the final downstream target of the major RhoA-mediated regulatory process in smooth muscle, will be determined with cryo-atomic force microscopy, a novel method developed by an investigator in this program (Project 2). These studies will also determine the structural bases of the interaction of the RhoA-regulated, smooth muscle myosin phosphatase with native, two-headed myosin molecules and the conformation of the myosin molecule during the cross bridge cycle that mediates contraction. The program will complete the development of a powerful structural method, energy filtered scanning transmission electron microscopy (STEM-EELS), that is designed to obtain compositional information at nanometer resolution about the cellular distribution of calcium, its movements and of other elements. This method will be used to quantitatively map calcium bound to cardiac and vascular smooth muscle cell membranes, and accumulated in mitochondria and other cell organelles and will rate these findings to the normal functions and abnormal effects of calcium. The cryo-atomic force microscope operating at ambient pressure and the intermediate high voltage, field emission gun- equipped STEM-EELS system and the very close interdisciplinary collaborations between structural biologists and cell physiologists are almost unique resources and features of this program. The high level of biomedical significance of this is derived from the involvement of the smooth muscle regulatory proteins in asthma and high blood pressure, and from the importance of calcium in the development of ventricular fibrillation, the most common cause of sudden cardiac death.
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