Liver FABP (fatty-acid-binding protein) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, lysophospholipids and bile acids. Liver FABP is also able to bind to anionic phospholipid vesicles under conditions of low ionic strength, and membrane binding results in the release of bound ligand. However, the molecular interactions involved in binding to the phospholipid interface and the mechanism of ligand release are not known. Ligand release could be due to a significant conformational change in the protein at the interface or interaction of a phospholipid molecule with the ligand-binding cavity of the protein resulting in ligand displacement. Two portal mutant proteins of liver FABP, L28W and M74W, have now been used to investigate the binding of liver FABP to anionic phospholipid vesicles, monitoring changes in fluorescence and also fluorescence quenching in the presence of brominated lipids. There is a large increase in fluorescence intensity when the L28W mutant protein binds to vesicles prepared from DOPG (dioleoyl-sn-phosphatidylglycerol), but a large decrease in fluorescence intensity when the M74W mutant binds to these vesicles. The Br4-phospholipid prepared by bromination of DOPG dramatically quenches both L28W and M74W, consistent with the close proximity of a fatty acyl chain to the tryptophan residues. The binding of liver FABP to DOPG vesicles is accompanied by only a minimal change in the CD spectrum. Overall, the results are consistent with a molecule of anionic phospholipid interacting with the central cavity of the liver FABP, possibly involving the phospholipid molecule in an extended conformation.

Liver fatty acid-binding protein (FABP) 1 is a member of a family of structurally related small (14 -15 kDa) cytosolic lipidbinding proteins that also include intestinal, heart (muscle), adipocyte, ileal, keratinocyte, and brain FABP (for recent reviews, see Refs. 1-7). The exact physiological functions of these proteins are unclear, although it is generally thought that they may have a potential role in the uptake and targeting of fatty acids to various intracellular organelles and metabolic pathways. All further sites of metabolism of long chain fatty acids in the cell involve membrane proteins. For a targeting role to operate, the FABP must interact with an intracellular structure such as a membrane interface or receptor/docking protein. Targeting could be modulated by the presence of bound fatty acid and be affected by the nature of the fatty acid such as chain length and the degree of unsaturation. In addition, FABP knock-out studies in mice have highlighted the physiological importance of acyl CoAs as ligands for liver FABP (8), and hence competition between these ligands and fatty acids could influence targeting of liver FABP.Evidence for membrane binding has been provided in the case of intestinal, muscle, and adipose FABP. Model fluorescence studies have led to the proposal for interaction-mediated transfer of long-chain fatty acids between the protein and a phospholipid (reviewed in Ref. 3). However, such a process was not observed to operate for liver FABP under the same assay conditions, and an aqueous phase diffusion mechanism was proposed, not requiring interaction of the protein with membrane surfaces (3). In contrast, using different assay conditions involving low ionic strength buffers we have reported the apparent binding of liver FABP to anionic vesicles, monitored as the release of the fluorescent fatty acid ligand (DAUDA) from the protein (9, 10).We have previously demonstrated that the binding of liver FABP to anionic vesicles involves nonspecific electrostatic interactions (9). As a result of studies using charge reversal mutagenesis, the cationic residues on the protein surface that make a significant contribution to such binding have been identified as Lys-31, Lys-36, and Lys-57 (10). These residues are strategically placed around the portal region of the proteins in positions of enhanced protein mobility. Our studies also highlighted the role of cationic residues in ligand binding at site 2 of liver FABP particularly ligands with more bulky anionic head groups such as acyl CoAs, lysophospholipids, and bile acids (11).To investigate further the effect of ligand and membrane binding on the conformation of liver FABP linked to a targeting role for this protein we have used a strategy of tryptophan insertion mutagenesis. Positions 28, 54, and 74 (see Fig. 1) were initially selected for mutagenesis to tryptophan because these positions are strategically placed surrounding the portal region. Fig. 1 is derived from the crystal structure with two bound oleates (12). The oleate at site 1 is buri...