Exp Eye Res 2001 Aug 01;732:257-64. doi: 10.1006/exer.2001.1031.

The functional unit of interphotoreceptor retinoid-binding protein (IRBP)--purification, characterization and preliminary crystallographic analysis.

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To define the relationship between the structure and function of interphotoreceptor retinoid-binding protein (IRBP) we seek to prepare crystals of IRBP suitable for X-ray crystallographic analysis. As recent studies suggest that each of IRBPs four homologous regions or modules possess ligand-binding activity, we here explore the feasibility of preparing crystals from an individual module. Xenopus laevis IRBP, which has a similar four-module structure as that of mammalian and avian IRBPs, was selected for these studies in view of the advantage of the Xenopus retina for cellular and transgenic approaches. In the present study we focused on the second module of Xenopus IRBP. This module was expressed as a thioredoxin/histidine-patch fusion protein to promote its soluble expression in Escherichia coli and subsequent purification. The ligand-binding properties of the fusion protein were determined by fluorescence spectroscopy. For the preparation of crystals, the module was enzymatically separated from the fusion tag. Crystals of the native and selenomethionine derivatized module were prepared by vapor diffusion in hanging drops. Module II of IRBP binds 1.57 +/- 0.041 and 1.49 +/- 0.15 equivalents of at all- trans retinol and 9-(9-anthroyloxy) stearic acid, respectively, with KDs in the 0.1 microM range. Crystals of this module had an elongated rectangular beam-like morphology. A complete dataset of a frozen selenomethionine crystal extending to 1.85 A resolution was collected. Focusing on the individual modules will likely provide an effective strategy to correlate biochemical and structural data to define the functional domains of IRBP. The quality and resolution of the data obtained suggests that it will be possible in the near future to solve the X-ray crystal structure of the IRBP modules.

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Species referenced: Xenopus laevis
Genes referenced: grb10 rbp3