Site-directed mutagenesis of rat cellular retinol-binding protein. Alteration in binding specificity resulting from mutation of glutamine 108 to arginine.
Cellular retinol-binding protein (CRBP) is a retinol-specific binding protein. A rat cDNA clone of CRBP was expressed in Escherichia coli. In order to determine amino acid residues in CRBP which may be important for the binding of all-trans-retinol, comparative model-building studies were performed in which strong sequence similarities were identified between CRBP and several other binding proteins. Based on this analysis, specific amino acids were predicted to be important in retinol binding, and these predictions were tested using the technique of site-directed mutagenesis to subtly alter the protein's structure and function. Specifically, site-directed mutagenesis was performed to alter the Gln-108 to Arg-108 (Q108R). Making use of fluorescence, Q108R was found to have a 3-fold lower affinity for all-trans-retinol, and the fine structure of the excitation spectrum of the Q108R.all-trans-retinol complex was also different than for the wild type.all-trans-retinol complex. The mutant bound 13-cis-retinol with an excitation spectrum identical to wild type bound to 13-cis-retinol, but with only one-half of the fluorescence intensity. In competition binding experiments, the Q108R mutant was found to have similar binding affinities for all-trans-retinol, all-trans-retinoic acid, 13-cis-retinoic acid, and retinal, while wild type CRBP was only able to bind to all-trans-retinol. Thus, altering a single amino acid in CRBP (Gln-108 to Arg-108) caused a significant change in the ligand binding specificity of the protein.
Stump
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DG
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Lloyd
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RS
Chytil
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F
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D051381:Rats; D012172:Retinaldehyde / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D054840:Retinol-Binding Proteins, Cellular; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014212:Tretinoin / Q000378:metabolism; D014801:Vitamin A / Q000378:metabolism
D009841:Oligonucleotides; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D014801:Vitamin A; D014212:Tretinoin; D012172:Retinaldehyde