Study of a fatty acid binding site of interphotoreceptor retinoid-binding protein using fluorescent fatty acids.
Interphotoreceptor retinoid-binding protein (IRBP) is a 140-kDa glycolipoprotein which constitutes about 70% of the soluble protein of the retinal interphotoreceptor matrix. Much is known concerning its role in the transport of retinoids between photoreceptor cells and pigment epithelium but little is known about its interactions with lipids. Here we have examined the physicochemical characteristics of a fatty acid binding site of IRBP using a set of fluorescent fatty acid analogs with an anthracene moiety attached at different positions along the hydrocarbon chain. The results show that fatty acids are bound in a hydrophobic environment as indicated by a blue shift in fluorescence maxima and by a increase in quantum yield of the bound ligand. There is a single specific fatty acid binding site for each molecule of IRBP with an apparent Kd = 3.6 x 10(-7) M. There is a nonradiative energy transfer from tryptophan residues to bound ligand. The interactions of IRBP and bound fatty acid are sensitive to denaturation by increasing concentrations of urea as judged by changes in nonradiative energy transfer efficiency and the quantum yield of bound probe. Quantum yields of bound fatty acid analogs varied with position of the fluorophore along the hydrocarbon chain and had the lowest values for the fluorophore located at the midpoint. Probing of the microenvironment of bound fluorophore with a quencher indicated a highly structured binding site.
Putilina
T
T
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
Sittenfeld
D
D
Chader
G J
GJ
Wiggert
B
B
D000818:Animals; D000873:Anthracenes / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004735:Energy Transfer; D005227:Fatty Acids / Q000378:metabolism*; D005456:Fluorescent Dyes; D010786:Photoreceptor Cells / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
D000873:Anthracenes; D005227:Fatty Acids; D005456:Fluorescent Dyes; D012177:Retinol-Binding Proteins; D014508:Urea