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

• Type of Material: Article
• Authors: Eleanore A. Gross, affiliation; Gui Ru Li, affiliation; Sarah E. Ruuska, affiliation; Jeffrey Boatright, Emory University; I. Saira Mian, University of California, Berkeley; John Nickerson, Emory University
• Language: English
• Date: 2000-04-07
• Publisher: Molecular Vision
• Publication Version: Final Published Version
• Copyright Statement: © 2000 Molecular Vision
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://www.molvis.org/molvis/v6/a7/
• Title of Journal or Parent Work: Molecular Vision
• ISSN: 1090-0535
• Volume: 2000
• Issue: 6
• Start Page: 40
• End Page: 50
• Grant/Funding Information: These studies were funded by NIH R01 EY10553 (to JMN), P30 EY06360, and T32 EY 07092; a Center grant from the Foundation Fighting Blindness; and an unrestricted grant to the Emory Eye Center from Research to Prevent Blindness; and the Director, Office of Science, Office of Basic Energy Sciences, U.S. Department of Energy, under Contract number DE-AC03-76SF0098 (to ISM).
• Research Categories: Health Sciences, Opthamology

Abstract

Purpose: The purpose of this study was to measure the effects of mutations on the retinol binding capability of human Repeat 1 of interphotoreceptor retinoid-binding protein (IRBP). First, we predicted important functional amino acids by several computer programs. We also noted the lack of shared functions between Tail-specific protease (Tsp) and IRBP, which bear sequence similarity, and this aided in predicting functional residues. We analyzed the effects of point substitutions on the retinol and fatty acid binding properties of Repeat 1 of human IRBP at 25 and 50 °C. Methods: To find residues critical to retinol binding that might affect function, a series of thirteen mutations were created by site-specific mutagenesis between positions 140 and 280 in Repeat 1 of human IRBP. These mutants were expressed, purified, and tested for binding properties. The conformations of the proteins were examined by circular dichroism (CD) scans. Results: Seven of the mutations exhibited reduced binding capacity, and five 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 those of Repeat 1. Only one of the mutations did not affect binding, folding, or expression when compare to wild type Repeat 1. Conclusions: Several IRBP mutants containing point mutations retained native structure but lost retinol binding function. The data suggest that retinol binding is affected by many different amino acid substitutions in or near a binding pocket. That even a single point substitution can profoundly affect binding without affecting overall conformation suggests that much of Domain B (from amino acid positions 80 to 300) is involved with ligand binding. This excludes three previously proposed IRBP-retinol binding mechanisms: (1) retinol binds to a small portion of the protein repeat, (2) retinol can bind to any hydrophobic patch in the protein, and (3) native conformation is not required for retinol binding to the repeat.