title: Fatty acid interactions with rat intestinal and liver fatty acid-binding proteins expressed in Escherichia coli. A comparative 13C NMR study.
abstract: Enterocytes in the small intestinal mucosa contain abundant quantities of two homologous cytosolic proteins known as intestinal and liver fatty acid-binding proteins (I- and L-FABP, respectively). To elucidate structure-function relationships for these proteins, the interactions between 13C-enriched palmitate and oleate and Escherichia coli-expressed rat I- and L-FABP were systematically compared using 13C NMR spectroscopy. NMR spectra of samples containing fatty acids (FA) and I-FABP at different molar ratios (all at pH 7.2 and 37 degrees C) exhibited a single carboxyl resonance corresponding to FA bound to I-FABP (181.4 ppm, peak I) and an additional carboxyl resonance corresponding to unbound FA in a bilayer phase (179.6 ppm). Peak I reached a maximum intensity corresponding to 1 mol of bound FA/mol of I-FABP under all sample conditions examined. NMR spectra for samples containing FA and L-FABP also exhibited a single carboxyl resonance corresponding to FA bound to L-FABP but at a different chemical shift value (182.2 ppm, peak L). Its maximum intensity varied depending on the physical state of the unbound FA (liquid crystalline or crystalline), the FA used (palmitate or oleate), and the sample pH. In the presence of a liquid crystalline (bilayer) phase, up to 1 (oleate) or 2 (palmitate) mol of FA were bound/mol of L-FABP, but in the presence of a crystalline phase (1:1 acid-soap), up to 3 mol of palmitate were bound/mol of L-FABP (all at pH 7.2). Peak I exhibited little or no ionization shift over a wide pH range (pH 3.0-11.0), and its chemical shift was unaffected by the ionization of Lys and His residues. Hence, the carboxylate group of FA bound to I-FABP was solvent inaccessible and most likely involved in an ion-pair electrostatic interaction with the delta-guanidinium moiety of an Arg residue. In contrast, peak L exhibited an ionization shift and an estimated apparent pKa value similar to that obtained for monomeric FA in water, suggesting that the carboxylate groups of FA bound to L-FABP were solvent accessible and located at or near the protein solvent interface. With decreasing pH, FA dissociated from L-FABP but not I-FABP, as monitored by NMR peak intensities. Concurrently, a large decrease in circular dichroism molar ellipticity was observed with L-FABP but not I-FABP. In conclusion, I-FABP and L-FABP are distinct with regards to their FA-binding stoichiometries, binding mechanisms, and sensitivity to pH.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Housman Medical Research Center, Department of Medicine, Boston University School of Medicine, Massachusetts 02118.
authors.forename: D P
authors.initials: DP
authors.lastname: Cistola
authors.forename: J C
authors.initials: JC
authors.lastname: Sacchettini
authors.forename: L J
authors.initials: LJ
authors.lastname: Banaszak
authors.forename: M T
authors.initials: MT
authors.lastname: Walsh
authors.forename: J I
authors.initials: JI
authors.lastname: Gordon
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers; D008099:Liver / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008962:Models, Theoretical; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009928:Organ Specificity; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D002247:Carbon Isotopes; D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D008051:Lipid Bilayers; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins