The aims of the research proposed are to understand in molecular detail and rearrangements that occur amongst the components of pulmonary surfactant as it is metabolized in the alveolar space. Previous studies by the applicant suggest that calcium-induced conformation transitions (C+ conformation) in SP-A may be a critical step in the lamellar body to tubular myelin transformation. Additional evidence suggests that at least part of the conformation transition occurs in the carbohydrate recognition domain of SP-A. The nature of the C= conformation in SP-A will be studied. Whether the conformation transition includes the exposure of a hydrophobic domain will be determined. Use will be made of hydrophobic interaction chromatography analysis of SP-A in the presence of calcium and its chelator EDTA and fluorescent hydrophobic probes labelling of the exposed domain. This domain of SP-A will be covalently labelled using carbene-generating photoreactive hydrophobic reagents in an attempt at determining its precise location. Existing antibodies specific for the C_ conformation in SP-A will be characterized by immunoblot analysis and ELISA. The ability of antibodies to inhibit calcium-dependent properties of SP-A, membrane aggregation and fusion, will be tested. The antibody epitope will be mapped on the C+ conformation of SP-A, using limited proteolysis and matching of the epitope against random peptides generated in a peptide library. The domain of SP-A defined by hydrophobic and epitope mapping will be tested for its functional importance by site-directed mutagenesis of the carbohydrate recognition domain (CRD) and full length SP-A cDNAs, using oligonucleotide-mediated techniques. The modified proteins will be tested for their ability to undergo calcium-induced conformation transitions, and promote membrane aggregation and fusion.
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