Purification of the full-length Xenopus interphotoreceptor retinoid binding protein and growth of diffraction-quality crystals.
OBJECTIVE
Interphotoreceptor retinol-binding protein (IRBP), composed of two or four homologous modules in tandem, plays an important role in retinoid trafficking between the retinal pigmented epithelium, photoreceptors, and Müller cells. The exact nature of this role is not yet clear. Attempts to purify the full-length retinal IRBP to homogeneity for crystallization purposes have largely been unsuccessful, owing primarily to instability and denaturation of the protein at high concentrations in aqueous media.


METHODS
A bacterial expression system was used for the production of the recombinant full-length four modules-containing Xenopus IRBP (xIRBP; 1197 amino acids; 131 kDa). An optimized purification strategy and the presence of molar excesses of a thiol-based reducing agent yielded highly pure xIRBP in a soluble, stable and active form, free of its fusion partner. Binding of all-trans retinol was characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer.


RESULTS
We grew the first diffraction-quality crystal of xIRBP. We have gathered diffraction data from these crystals to 2.46 A resolution, sufficient to yield an atomic model of the tertiary structure of IRBP. Retinol-binding results determined by fluorescence spectroscopy show roughly one retinol-binding site per polypeptide chain.


CONCLUSIONS
The binding stoichiometry taken together with modeling data suggest that not all modules are functionally equivalent. Determination of the full-length IRBP structure will be a significant breakthrough in understanding the functional roles of IRBP in the visual cycle. The advances presented here will not only lead to the structure of the full-length IRBP, but will allow us to understand how the modules interact in the function of IRBP. Furthermore, these studies will allow characterization of the ligand-binding site(s) with bound ligand(s).
Ghosh
Debashis
D
Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203, USA. ghosh@hwi.buffalo.edu
Griswold
Jennifer B
JB
Bevilacqua
Thomas
T
Gonzalez-Fernandez
Federico
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D003460:Crystallization; D004355:Drug Stability; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008024:Ligands; D008433:Mathematics; D008956:Models, Chemical; D015394:Molecular Structure; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014801:Vitamin A / Q000378:metabolism; D014961:X-Ray Diffraction; D014981:Xenopus / Q000378:metabolism*; D000099214:Retinol-Binding Proteins, Interstitial
D005136:Eye Proteins; D008024:Ligands; D012177:Retinol-Binding Proteins; D014801:Vitamin A; D000099214:Retinol-Binding Proteins, Interstitial