DESCRIPTION (Adapted from abstract): The long-term goal of this project is to understand the molecular mechanism of thin filament linked smooth muscle regulation in atomic detail. Malfunction in smooth muscle contraction may contribute to diseases such as asthma and hypertension. Two independent mechanisms, respectively responsible for switch and modulatory fine-tuning, seem to regulate smooth muscle contraction. The primary mechanism is switched on by phosphorylation of the myosin regulatory light chain. The other, less well-established mechanism, involves caldesmon , a protein found in association with the actin thin filaments in smooth muscle cells. Caldesmon is a flexible and elongated protein and its atomic structure is still unknown. The C-terminal domain of caldesmon, however, is more structured and is responsible for nearly all the functional interactions of the molecule including binding to actin, tropomyosin, and Ca2+-dependent regulatory proteins (such as calmodulin and S100 proteins). To understand caldesmon regulatory function there is an urgent need to know the atomic structure of this domain and its interactions with other modulatory proteins. The specific aims of this proposal are 1 ) To determine the crystal structures of the complexes between Ca 2+- calmodulin and two caldesmon target peptides, 2) To determine the atomic structure of a complex between Ca2+-calmodulin and a proteolytic fragment from the C-terminal domain of caldesmon, 3) To determine the apo and Ca2+-bound crystal structures of human calcyclin (a S100 protein), 4) To crystallize and determine the structure of a complex between Ca2+-calcyclin and a caldesmon proteolytic fragment. The primary tool will be x-ray crystallography, but other techniques will be used as well for the preparation and characterization of the proteins. Accomplishment of these specific aims will considerably advance our understanding of thin filament linked Ca2+-modulated regulation of smooth muscle, and will unveil the differences and similarities in target recognition by calmodulin and S100 proteins.
NIH ExPorter
NIH ExPorter