Retinoic acid, a hormonally active metabolite of vitamin A, has profound effects on cell growth, differentiation, and morphogenesis. Two kinds of proteins are found to be involved in the functions of retinoic acid: nuclear retinoic acid receptors (RARs and RXRs) and cellular retinoic acid binding proteins (CRABPs). RARs and RXRs are retinoic acid dependent transcriptional factors that regulate expression of target genes. CRABPs are thought to play important roles in transport and metabolism of retinoic acid and may modulate the concentrations of free retinoic acid in target cells and tissues. Although great advances have been made in our understanding of the roles of retinoic acid in recent years, little is known about how CRABPs, RARs and RXRs interact with retinoic acid and what are the conformational changes upon binding of the ligand. The long-term goal of the proposed project is to elucidate the interactions between CRABPs and retinoic acid and the effects of ligand binding on the conformations of the proteins. Our model system is human CRABP II expressed in E. coli. A combination of site-directed mutagenesis, equilibrium binding and biophysical methods will be used.
The specific aims of the proposed project are: (1) to identify the amino acid residues involved in binding of retinoic acid by site-directed mutagenesis; (2) to analyze quantitatively the ligand binding properties of the site-directed mutants; (3) to assess the effects of mutations on the structure and stability of human CRABP II by biophysical methods; (4) to characterize the solution structures of human CRABP II and its complex with retinoic acid by NMR. As a long-term plan, we are also interested in comparing the ligand binding properties CRABP II with those of CRABP I, RARs and RXRs.
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