STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF RECOMBINANT HUMAN CELLULAR RETINALDEHYDE-BINDING PROTEIN

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the ret inal pigment epithelium (RPE) and Muller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regen eration. The protein carries 11-cis-retinal and/or 11-cis-retinol as e ndogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autoso mal recessive retinitis pigmentosa. CRALBP is also present in brain wi thout endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde -binding protein (rCRALBP) has been over expressed as non-fusion and f usion proteins in Escherichia coli from pET3a and pET19b vectors, resp ectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3 -8 mg per liter of bacterial culture. Liquid chromatography electrospr ay mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structur e and mass. Circular dichroism, retinoid HPLC, UV-visible absorption s pectroscopy, and solution state F-19-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retino id-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exh ibited by bovine and human CRALBP when complexed with 9-cis-retinal. T hese recombinant preparations provide valid models for human CRALBP st ructure-function studies.