Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding. Study holds ProTherm entries: 12300, 12301, 12302, 12303, 12304, 12305, 12306 Extra Details: beta-barrel; protein engineering; protein folding; electrostatic interactions;,protein-ligand interaction
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:43 p.m.
|Number of data points||10|
|Proteins||Cellular retinoic acid-binding protein 1 ; Cellular retinoic acid-binding protein 1|
|Assays/Quantities/Protocols||Experimental Assay: dG ; Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence PNFAGTWKMRSSENFDELLKALGVNAMLRKVAVAAASKPHVEIRQDGDQFYIKTSTTVRTTEINFKVGEGFEEETVDGRKCRSLPTWENENKIHCTQTLLEGDGPKTYWTRELANDELILTFGADDVVCTRIYVRE|