Interactions of fatty acids with neutral fatty-acid-binding protein from bovine liver.
Hepatic-type fatty-acid-binding protein (hFABP) from the cytosol of bovine liver is a 14.4-kDa neutral protein with a blocked N-terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 microM and 2.15 microM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12% alpha-helix, 45% beta-structure, 15% beta-turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding. Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as 1H-NMR analysis of His-C2 and His-C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyrocarbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%. From 1H-NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/protein interaction.
Schulenberg-Schell
H
H
Institut für Biochemie, Universität Münster, Federal Republic of Germany.
Schäfer
P
P
Keuper
H J
HJ
Stanislawski
B
B
Hoffmann
E
E
Rüterjans
H
H
Spener
F
F
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009363:Neoplasm Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D011487:Protein Conformation
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009829:Oleic Acids; D019301:Oleic Acid