Abstract
Publisher Summary Because of the low solubility of hydrophobic molecules in water, it has been difficult to measure the binding properties of the site-directed mutants of the proteins that interact with hydrophobic ligands, such as cellular retinoic acid binding proteins (CRABPs). This has greatly hampered the studies of the quantitative structure–function relationships of these important proteins. Retinoic acid (RA), a hormonally active metabolite of vitamin A, has profound effects on cell growth, differentiation, and morphogenesis. Two types of proteins have been found to bind RA: nuclear retinoic acid receptors (RARs and RXRs) and CRABPs. It appears that the two isoforms may have distinct functions. Four conserved residues (Arg-Ul, Leu-121, Arg-132, and Tyr-134 in CRABP-n) line at the bottom of the RA binding pockets of CRABPs and interact with the carboxyl group of RA. However, the affinities of these mutants for RA have not been quantitatively determined, because the current RA binding assays are inapplicable to mutants with greatly decreased affinity for RA. A novel competitive binding assay has been developed for measuring the dissociation constants of the site-directed mutants of CRABPs. This method has been used to evaluate the contribution of Leu-121 of CRABP-II to binding of RA in conjunction with site-directed mutagenesis and NMR. The results show that Leu-121 is also important for binding of RA and contributes to the binding energy by ∼ 1.4 kcal/mol.
Published Version
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