Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants.
Rat liver fatty-acid-binding protein (FABP) does not contain tryptophan. Three mutant proteins have been produced in which a single tryptophan residue has been inserted by site-directed mutagenesis at positions 3 (F3W), 18 (F18W) and 69 (C69W). These tryptophans have been strategically located in order to provide fluorescent reporter groups to study the binding and structural characteristics of rat liver FABP. Two fluorescent fatty acid analogues, DAUDA (11-[(5-dimethylaminonaphthalene-1- sulphonyl)amino]undecanoic acid) and 3-[p-(6-phenyl)-hexa-1,3,5-trienyl]phenylpropionic acid, showed no significant difference in binding affinities for the different mutant proteins, although maximum fluorescence values were decreased for F3W and increased for C69W. These findings were confirmed by studies of DAUDA displacement by oleate. Protein-denaturation studies in the presence of urea indicated subtle differences for the three mutants which could be explained by multiple unfolding pathways. Fatty acid binding increased tryptophan fluorescence emission in the case of the F18W protein, but had no effect on the F3W and C69W proteins. Fluorescence quenching studies with 2-bromopalmitate showed that a fatty acid carboxylate is close to the tryptophan in the F18W protein. Energy-transfer studies showed that the fluorescent moiety of DAUDA is equidistant from the three mutated amino acids and is bound within the beta-clam solvent cavity of liver FABP. This interpretation of the fluorescence quenching and energy-transfer data supports the difference in ligand orientation between intestinal and liver FABP observed in previous studies.
Department of Biochemistry, University of Southampton, U.K.
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Thumser
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Wilton
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004735:Energy Transfer; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D005456:Fluorescent Dyes; D008099:Liver / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011489:Protein Denaturation; D051381:Rats; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D014364:Tryptophan; D014508:Urea