Effects of dispersed point substitutions in Repeat 1 of human interphotoreceptor retinoid binding protein (IRBP)

PURPOSE: The purpose of this study was to measure the effects of mutations on the retinol binding capability of human Repeat 1 of interphotoreceptor r etinoid-binding protein (IRBP). First, we predicted important functional am ino acids by several computer programs. We also noted the lack of shared fu nctions between Tail-specific protease (Tsp) and IRBP, which bear sequence similarity, and this aided in predicting functional residues. We analyzed t he effects of point substitutions on the retinol and fatty acid binding pro perties of Repeat 1 of human IRBP at 25 and 50 degrees C. METHODS: To find residues critical to retinol binding that might affect fun ction, a series of thirteen mutations were created by site-specific mutagen esis between positions 140 and 280 in Repeat 1 of human IRBP. These mutants were expressed, purified, and tested for binding properties. The conformat ions of the proteins were examined by circular dichroism (CD) scans. RESULTS: Seven of the mutations exhibited reduced binding capacity, and fiv e were not expressed at high enough levels to assess binding activity. Four of the mutants were purified, and their CD scans were very similar to thos e of Repeat 1. Only one of the mutations did not affect binding, folding, o r expression when compare to wild type Repeat 1. CONCLUSIONS: Several IRBP mutants containing point mutations retained nativ e structure but lost retinol binding function. The data suggest that retino l binding is affected by many different amino acid substitutions in or near a binding pocket. That even a single point substitution can profoundly aff ect binding without affecting overall conformation suggests that much of Do main B (from amino acid positions 80 to 300) is involved with ligand bindin g. This excludes three previously proposed IRBP-retinol binding mechanisms: (1) retinol binds to a small portion of the protein repeat, (2) retinol ca n bind to any hydrophobic patch in the protein, and (3) native conformation is not required for retinol binding to the repeat.