SOLUBLE EXPRESSION IN ESCHERICHIA-COLI OF A FUNCTIONAL INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN MODULE FUSED TO THIOREDOXIN - CORRELATION OF VITAMIN-A BINDING REGIONS WITH CONSERVED DOMAINS OF C-TERMINAL PROCESSING PROTEASES

The exchange of all-trans retinol and 11-cis retinal between the photo receptors and retinal pigmented epithelium is mediated by interphotore ceptor retinoid-binding protein (IRBP). IRBP contains binding sites fo r retinoids, docosahexaenoic acid and probably cell surface and matrix receptors. IRBP arose through the quadruplication of an ancient prote in, represented by its carboxy-terminal module (module 4 in amphibians and mammals). Module 4 has retinol binding activity and is composed o f regions coded for by each of IRBP's four exons. Determining the func tion of the exons has been hampered by insoluble expression of module 4 in Escherichia coli. Here, we found that module 4 of Xenopus IRBP (X 4IRBP), as well as its exon segments, can be expressed in a soluble fo rm as thioredoxin fusion proteins. The recombinant proteins were purif ied by ion exchange and arsenical-based affinity chromatography. Liqui d chromatography/mass spectrometry confirmed that the sequence of X4IR BP is correct. All-trans retinol binding was characterized by monitori ng enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol. Retinol bo und to X4IRBP at 2.20+/-0.29. One of the two sites was localized to Ex ons(2 + 3) and had a K-D = 0.26+/-0.13 mu M. This site, which supporte d protein quenching and energy transfer, probably contains at least on e of the two conserved tryptophans present in this segment. The second site was localized to Exon 4. This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had a K-D = 1.94+/-0.20 mu M. Exon 1 had no retinol binding activity. The location of the retinol binding regions correlated with the distri bution of domains conserved between IRBPs and the newly recognized fam ily of C-terminal processing proteases (CtpAs), proteins which bind an d cleave non-polar carboxy termini. (C) 1998 Academic Press Limited.