The purpose of this research is to understand the molecular basis of steroid-protein interaction, a biochemical process functioning in steroid hormone action and steroid metabolism. The Sex Steroid-Binding Protein, SBP, of human (hSBP) and rabbit (rSBP) plasma have been chosen as protein models to accomplish this objective. SBP is the sole noncatalytic sex steroid-binding protein in the plasma of most species, including humans, which specifically binds testosterone (T), dihydrotestosterone (DHT), and 17beta-estradiol (E2) with high affinity. Since rabbit SBP only binds the androgens, both proteins will be isolated and compared to reveal the molecular determinants responsible for distinguishing androgens from estrogens. The methods of chemistry, protein chemistry, molecular biology, and X-ray diffraction will be used to complete the biochemical characterization of both proteins, including their steroid-binding sites and solution of the three-dimensional structure. The project is composed of three specific aims: (1) Specific Aim 1 is to complete the physicochemical characterization and undertake solution of the three- dimensional structure. (2) Specific Aim 2 is to characterize the steroid- binding site by affinity labeling and site-directed mutagenesis to identify amino acid side-chains that function in the steroid-binding process. (3) Specific Aim 3 is to express SBP in bacteria and insect cells to produce large amounts of deglycosylated recombinant SBP for crystallization trials and structure analysis. It was recently found that deglycosylated SBP is fully-active and is easier to crystallize. The long-term goal of this research is to describe, in as much detail as possible, the structure of a noncatalytic steroid-binding protein in order to understand the molecular basis of steroid-protein interaction. The structural information obtained in these studies should apply to other steroid-binding proteins since they all recognize the same basic steroid nucleus. The results obtained should stimulate research on the design of synthetic steroids or other drugs that can compete with natural hormones and thus could provide opportunities for developing therapeutic approaches in the treatment of steroid-dependent diseases. Furthermore, the results will have an important impact on the recent discovery of the SBP receptor which has been implicated in the specific uptake of sex steroid hormones into target cells.
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