Truncational mutagenesis of the fourth module of xenopus irbp

Interphotoreceptor retinoid-binding protein (IRBP) is composed of multiple repeats or modules (4 in amphibians and mammals, 2 in teleosts) of about 300 amino acids The fourth (N-terminal) repeat contains a single binding site for all-trans retinol (Baer et at, Curr Eye Res 13:391-400,1994) Purpose. Define regions within the fourth module that are critical to ligand binding. Methods. The amino acid sequences of the N-terminal modules of human, bovine, Xenopus, goldfish, and zebrafish IRBPs were aligned From this alignment, we selected three regions to express: fragment A (residues 1-87), fragment B (81-201), and fragment C (195-306). These fragments as well as the whole fourth module were expressed in soluble form as thioredoxin fusion proteins and purified by a combination of affinity and classical Chromatographic approaches The binding of all-trans retinol to all four proteins was determined by fluorometric titration Results. The fourth module of Xenopus IRBP had 0.9 binding sites (N) with a Kd = 0 4 uM Most of the binding activity was found in fragment B (central region). This fragment had N = 0 9 with Kd = 0.2 (aM. Fragment A had little binding activity (10% of fragment B). Fragment C had some activity (25% of fragment B). Conclusions. The thioredoxin expression is a useful strategy to express soluble IRBP in E coli. Our data suggest that all-trans retinol binding is primarily restricted to a phylogenetically conserved hydrophobic domain in the central region of the fourth module of IRBP. NIH EY09412 and NTH GM 07267 None.