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Purification of the full-length Xenopus interphotoreceptor retinoid binding protein and growth of diffraction-quality crystals.
Ghosh D
,
Griswold JB
,
Bevilacqua T
,
Gonzalez-Fernandez F
.
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PURPOSE: 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).
Figure 1. Gel electrophoresis (SDSâPAGE; silver stained) analysis of the purified four-module containing Xenopus interphotoreceptor retinol-binding protein. The major band corresponds to monomeric Xenopus interphotoreceptor retinol-binding protein (xIRBP) molecular weight of 131 kDa. The minor higher molecular weight band represents dimeric form of the protein, which may or may not be an artifact of the analysis itself.
Figure 2. Fluorescence titrations are shown of Xenopus interphotoreceptor retinol-binding protein (xIRBP) binding all-trans retinol. The concentration of xIRBP was 0.89 μM in each panel. A: The titration of IRBP with all-trans retinol in this panel is followed by monitoring the increase in retinol fluorescence compared to a fluorescence matched solution of N-acetyl-L-tryptophanamide (excitation: 330 nm; emission: 480 nm). B: The titration shown follows the quenching of intrinsic protein fluorescence by bound retinol (excitation: 280 nm; emission: 340 nm). C: The titration follows ligand binding by monitoring energy transfer (excitation: 280 nm; emission: 480 nm).
Figure 3. Photomicrographs of Xenopus interphotoreceptor retinol-binding protein crystals grown in the presence of oleic acid. The crystals were typically about 0.2 mm in the longest dimension.
Figure 4. An X-ray diffraction pattern from a Xenopus interphotoreceptor retinol-binding protein crystal recorded at the A-1 station of the Cornell High Energy Synchrotron Source. The detector was at a distance 250 mm; the wavelength, the oscillation angle and the exposure time were 0.978 à , 1°, and 15 s, respectively. The right upper corner segment is shown in higher contrast to demonstrate the limiting resolution of diffraction of ~2.45 à .
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