Nmr and mutational analysis of retinoid-binding in the cellular retinaldehyde-binding protein (cralbp)

Purpose. To identify amino acid residues in CRALBP associated with the retinoid binding pocket. CRALBP may play a regulatory role in the visual cycle regeneration of 11-o/s-retinaldehyde (11-c/s-Ral). Methods. Recombinant human CRALBP was labeled with "C-methionine, 5-fluorotrytophan or uniformly with "N by biosynthetic incorporation. LCESMS was used to evaluate isotope incorporation. Solution state 13C, 19F and 15N NMR analyses of CRALBP were used to demonstrate local conformational changes associated with Met and Trp residues before and after bleaching (i.e., with and without bound 11-cis-Ral). Six rCRALBP Trp and Met mutants were produced in bacteria and assayed for retinoid binding. CRALBP exhibits greater affinity for 11 -cis than 9-cis-Ral, therefore 9-cis-Ral is a more sensitive probe for the structural integrity of the binding pocket. Results. Preliminary results indicate rCRALBP mutants W165F, M225A and W244F bind 11-cis-Ral but not 9-cis-Ral, mutant M208A binds both 11-cis and 9-cis-Ral and mutant M222A binds neither 11-cis or 9-cis-Ral. Mutant M158A soluble protein is expressed at lower levels than the other mutants. Further retinoid binding studies are in progress. Conclusions. CRALBP residues W165, M222, M225, W244 appear to be components of the retinoid-binding pocket. NMR analysis will be used to elucidate conformational changes associated with retinoid binding.

Duke Scholars

Author Ronald Venters Radiology