Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.
Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without 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 fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. 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 electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-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 retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.
Crabb
J W
JW
Adirondack Biomedical Research Institute, Lake Placid, New York 12946, USA. jcrabb@cell-science.org
Carlson
A
A
Chen
Y
Y
Goldflam
S
S
Intres
R
R
West
K A
KA
Hulmes
J D
JD
Kapron
J T
JT
Luck
L A
LA
Horwitz
J
J
Bok
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006358:Hot Temperature; D006801:Humans; D008027:Light; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D012176:Retinoids / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012176:Retinoids