Calponin (CaN) has been implicated in thin filament-based regulation of smooth muscle contraction by virtue of its similarities to skeletal muscle troponin I and troponin T. Little, however, is known about the structure and function of CaN. The objectives of this project are to gain information on the structural features of CaN and how it interacts with other smooth muscle proteins in the thin filament. A combination of site- directed mutagenesis, photocrosslinking and fluorescence spectroscopic techniques will be used. It is anticipated that such information will allow us to deduce the role that CaN plays in smooth muscle regulation and how it functions in this role.
The specific aims of this project are as follows: (1) To determine the gross conformation of CaN. The secondary structure will be analyzed by circular dichroism (CD) spectroscopy, and its size and shape by electron microscopy and analytical ultracentrifugation. (2) To determine the proximity relationships between specific sites in CaN. Mutants containing pairs of Cys's at defined locations will be generated by site-directed mutagenesis, and the distances between them measured by resonance energy transfer (RET). (3) To characterize the properties of fragments corresponding to various regions of CaN. Fragments of CaN will be generated by genetic engineering techniques, and their properties such as secondary and tertiary structure, and modes of interaction with other thin filament proteins analyzed. (4) To identify and characterize sites of interaction with other smooth muscle thin filament proteins. Mutants of CaN containing single Cys's at putative interaction sites will be generated and labeled with photoactivable crosslinkers. Photocrosslinking with target proteins will be achieved by ultraviolet irradiation, and crosslinked products will be subjected to peptide and amino acid analyses to identify the amino acid residues that are involved. (5) To determine the parameters of interaction between CaN and its target proteins. Interaction parameters (stoichiometry and affinity constant) will be measured by fluorescence and fluorescence polarization techniques using single Cys mutants of CaN. (6) To determine the spatial arrangement of CaN in relationship to other proteins in the smooth muscle thin filament. Using single Cys mutants of CaN, the distances between various sites in CaN and various """"""""landmark"""""""" sites in other thin filament proteins will be measured by RET. Such distance information will be used to deduce how CaN is """"""""positioned"""""""" in the smooth muscle thin filament. Information from this project will contribute towards the understanding of normal smooth muscle function, and may eventually provide clues to the prevention and cure of smooth muscle related diseases.
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