title: Arginine to glutamine substitutions in the fourth module of Xenopus interphotoreceptor retinoid-binding protein.
abstract: OBJECTIVE
  Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of approximately 300 amino acid residues. All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions [Baer et al, Exp Eye Res 1998; 66:249-262]. Here we investigate the role of highly conserved arginines contained in these regions.
  
  
  METHODS
  To study the arginines in an individual module without the interference of ligand-binding activity elsewhere in the protein, we expressed in E. coli the fourth module of Xenopus IRBP by itself as a soluble thioredoxin fusion protein (X4IRBP). Arginines 1005, 1041, 1073 and 1122 were separately replaced by glutamine using PCR overlap extension mutagenesis. The glutamine substitutions were confirmed by liquid chromatography-tandem mass spectrometry. The binding of all-trans retinol and 9-(9-anthroyloxy)stearic acid (9-AS) to X4IRBP and each of the mutants was evaluated by fluorescence spectroscopy. Binding was followed by monitoring the enhancement of ligand fluorescence and the quenching of protein endogenous fluorescence. The ability of the recombinant proteins to protect all-trans retinol from oxidative degradation was evaluated by monitoring absorbance at 325 nm over time.
  
  
  RESULTS
  The substitution of Gln for Arg1005 about doubled the amount of ligand necessary to attain saturation and about doubled the level of fluorescence enhancement obtained at saturation for both all-trans retinol and 9-AS. Although there was not a significant change in the Kd, the substitution increased the calculated number of binding sites (N) from approximately 2 to approximately 4 per polypeptide. The other Arg->Gln mutants did not significantly change the Kd or N. None of the mutations compromised the ability of the module to protect all-trans retinol from degradation.
  
  
  CONCLUSIONS
  Our data suggest that the function of the conserved arginines in IRBP is fundamentally different from that of other retinoid-binding proteins. These residues do not appear to play a role in defining the specificity of the ligand-binding domain. Rather, Arg1005 appears to play a role in defining the capacity of the domain. Our data suggest that the binding site consists of a single hydrophobic cavity promiscuous for fatty acids and all-trans retinol.
authors.affiliation: Graduate Program in Neuroscience, University of Virginia Health Sciences Center, Charlottesville, VA 22906-0009, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Baer
authors.forename: E E
authors.initials: EE
authors.lastname: Van Niel
authors.forename: J W
authors.initials: JW
authors.lastname: Cronk
authors.forename: M T
authors.initials: MT
authors.lastname: Kinter
authors.forename: N E
authors.initials: NE
authors.lastname: Sherman
authors.forename: M S
authors.initials: MS
authors.lastname: Braiman
authors.forename: F
authors.initials: F
authors.lastname: Gonzalez-Fernandez
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D003001:Cloning, Molecular; D004926:Escherichia coli; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D015870:Gene Expression; D005973:Glutamine; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins; D012177:Retinol-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D013229:Stearic Acids / Q000493:pharmacokinetics; D014801:Vitamin A / Q000493:pharmacokinetics; D014981:Xenopus; D000099214:Retinol-Binding Proteins, Interstitial
chemical_list: D001120:Arginine; D005136:Eye Proteins; D005973:Glutamine; D011993:Recombinant Fusion Proteins; D012177:Retinol-Binding Proteins; D013229:Stearic Acids; D014801:Vitamin A; D000099214:Retinol-Binding Proteins, Interstitial; C065154:9-(9-anthroyloxy)stearic acid