Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids.
Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed.
Wang
Hsin
H
Department of Chemistry, CUNY Graduate Center and College of Staten Island, Staten Island, New York 10314, USA.
He
Yan
Y
Kroenke
Christopher D
CD
Kodukula
Sarala
S
Storch
Judith
J
Palmer
Arthur G
AG
Stark
Ruth E
RE
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002247:Carbon Isotopes / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism; D019308:Palmitic Acid / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D011869:Radioligand Assay; D051381:Rats; D017186:Titrimetry
D002247:Carbon Isotopes; D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D008024:Ligands; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011522:Protons; D019301:Oleic Acid; D019308:Palmitic Acid