The integrity of the alpha-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes

Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes.     

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.     

Fatty acid transfer from intestinal fatty acid binding protein to membranes: electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex", during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.     

Two fatty acid‐binding proteins expressed in the intestine interact differently with endocannabinoids

Two different members of the fatty acid‐binding protein (FABP) family are found in enterocyte cells of the gastrointestinal system, namely liver‐type and intestinal fatty acid‐binding proteins (LFABP and IFABP, also called FABP1 and FABP2, respectively). Striking phenotypic differences have been observed in knockout mice for either protein, for example, high fat‐fed IFABP‐null mice remained lean, whereas LFABP‐null mice were obese, correlating with differences in food intake. This finding prompted us to investigate the role each protein plays in directing the specificity of binding to ligands involved in appetite regulation, such as fatty acid ethanolamides and related endocannabinoids. We determined the binding affinities for nine structurally related ligands using a fluorescence competition assay, revealing tighter binding to IFABP than LFABP for all ligands tested. We found that the head group of the ligand had more impact on binding affinity than the alkyl chain, with the strongest binding observed for the carboxyl group, followed by the amide, and then the glycerol ester. These trends were confirmed using two‐dimensional ¹H–¹⁵N nuclear magnetic resonance (NMR) to monitor chemical shift perturbation of the protein backbone resonances upon titration with ligand. Interestingly, the NMR data revealed that different residues of IFABP were involved in the coordination of endocannabinoids than those implicated for fatty acids, whereas the same residues of LFABP were involved for both classes of ligand. In addition, we identified residues that are uniquely affected by binding of all types of ligand to IFABP, suggesting a rationale for its tighter binding affinity compared with LFABP.     

Similar mechanisms of fatty acid transfer from human anal rodent fatty acid-binding proteins to membranes: Liver,intestine, heart muscle,and adipose tissue FABPs

The mammalian fatty acid-binding proteins (FABPs) are thought to be important for the transport and metabolism of fatty acids in numerous cell types. The transfer of FA from different members of the FABP family to membranes has been shown to occur by two distinct mechanisms, an aqueous diffusion-based mechanism and a collisional mechanism, wherein the FABP interacts directly with membrane acceptors. Much of the work that underlies this concept comes from efforts using rodent FABPs. Given the increasing awareness of links between FABPs and several chronic diseases in humans, it was important to establish the mechanisms of FA transfer for human FABPs. In the present studies, we examined the rate and mechanism of fatty acid transfer from four pairs of human and rodent (rat or mouse, as specified) FABPs: hLFABP and rLFABP, hIFABP and rIFABP, hHFABP and rHFABP, and hAFABP and mAFABP. In the case of human IFABP, both the Ala⁵⁴ and Thr⁵⁴ forms were examined. The results show clearly that for all FABPs examined, the mechanisms of ligand transfer observed for rodent proteins hold true for their human counterparts. Moreover, it appears that the Ala to Thr substitution at residue 54 of the human IFABP does not alter the fundamental mechanism of ligand transfer to membranes, but nevertheless causes a consistent decrease in the rate of transfer.     

Deletion of the helical motif in the intestinal fatty acid-binding protein reduces its interactions with membrane monolayers: Brewster angle microscopy, IR reflection-absorption spectroscopy, and surface pressure studies

Intestinal fatty acid binding protein (IFABP) appears to interact directly with membranes during fatty acid transfer [Hsu, K. T., and Storch, J. (1996) J. Biol. Chem. 271, 13317-13323]. The largely alpha-helical "portal" domain of IFABP was critical for these protein--membrane interactions. In the present studies, the binding of IFABP and a helixless variant of IFABP (IFABP-HL) to acidic monolayers of 1,2-dimyristoylphosphatidic acid (DMPA) has been monitored by surface pressure measurements, Brewster angle microscopy (BAM), and infrared reflection-absorption spectroscopy (IRRAS). Protein adsorption to DMPA exhibited a two phase kinetic process consisting of an initial slow phase, arising from protein binding to the monolayer and/or direct interfacial adsorption, and a more rapid phase that parallels formation of lipid-containing domains. IFABP exhibited more rapid changes in both phases than IFABP-HL. The second phase was absent when IFABP interacted with zwitterionic monolayers of 1,2-dipalmitoylphosphatidylcholine, revealing the important role of electrostatics at this stage. BAM images of DMPA monolayers with either protein revealed the formation of domains leading eventually to rigid films. Domains of DMPA/IFABP-HL formed more slowly and were less rigid than with the wild-type protein. Overall, the IRRAS studies revealed a protein-induced conformational ordering of the lipid acyl chains with a substantially stronger ordering effect induced by IFABP. The physical measurements thus suggested differing degrees of direct interaction between the proteins and DMPA monolayers with the IFABP/DMPA interaction being somewhat stronger. These data provide a molecular structure rationale for previous kinetic measurements indicating that the helical domain is essential for a collision-based mechanism of fatty acid transfer to phospholipid membranes [Corsico, B., Cistola, D. P., Frieden, C. and Storch, J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 12174-12178].     

Modeling fatty acid delivery from intestinal fatty acid binding protein to a membrane

Intestinal fatty acid binding protein (IFABP) interacts with biological membranes and delivers fatty acid (FA) into them via a collisional mechanism. However, the membrane-bound structure of the protein and the pathway of FA transfer are not precisely known. We used molecular dynamics (MD) simulations with an implicit membrane model to determine the optimal orientation of apo- and holo-IFABP (bound with palmitate) on an anionic membrane. In this orientation, the helical portal region, delimited by the alphaII helix and the betaC-betaD and betaE-betaF turns, is oriented toward the membrane whereas the putative beta-strand portal, delimited by the betaB-betaC, betaF-betaG, betaH-betaI turns and the N terminus, is exposed to solvent. Starting from the MD structure of holo-IFABP in the optimal orientation relative to the membrane, we examined the release of palmitate via both pathways. Although the domains can widen enough to allow the passage of palmitate, fatty acid release through the helical portal region incurs smaller conformational changes and a lower energetic cost.     

Role of the helical domain in fatty acid transfer from adipocyte and heart fatty acid-binding proteins to membranes: analysis of chimeric proteins.

The adipocyte and heart fatty acid-binding proteins (A- and HFABP) are members of a lipid-binding protein family with a beta-barrel body capped by a small helix-turn-helix motif. Both proteins are hypothesized to transport fatty acid (FA) to phospholipid membranes through a collisional process. Previously, we suggested that the helical domain is particularly important for the electrostatic interactions involved in this transfer mechanism (Herr, F. M., Aronson, J., and Storch, J. (1996) Biochemistry 35, 1296-1303; and Liou, H.-L., and Storch, J. (2001) Biochemistry 40, 6475-6485). Despite their using qualitatively similar FA transfer mechanisms, differences in absolute transfer rates as well as regulation of transfer from AFABP versus HFABP, prompted us to consider the structural determinants that underlie these functional disparities. To determine the specific elements underlying the functional differences between AFABP and HFABP in FA transfer, two pairs of chimeric proteins were generated. The first and second pairs had the entire helical domain and the first alpha-helix exchanged between A- and HFABP, respectively. The transfer rates of anthroyloxy-labeled fatty acid from proteins to small unilamellar vesicles were compared with the wild type AFABP and HFABP. The results suggest that the alphaII-helix is important in determining the absolute FA transfer rates. Furthermore, the alphaI-helix appears to be particularly important in regulating protein sensitivity to the negative charge of membranes. The alphaI-helix of HFABP and the alphaII-helix of AFABP increased the sensitivity to anionic vesicles; the alphaI-helix of AFABP and alphaII-helix of HFABP decreased the sensitivity. The differential sensitivities to negative charge, as well as differential absolute rates of FA transfer, may help these two proteins to function uniquely in their respective cell types.     

Direct Comparison of Mice Null for Liver or Intestinal Fatty Acid-binding Proteins Reveals Highly Divergent Phenotypic Responses to High Fat Feeding

The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP^−/− and LFABP^−/− mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP^−/− mice, whereas LFABP^−/− mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP^−/− mice fed high fat diets became obese relative to WT, whereas IFABP^−/− mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP^−/− mice preferentially utilizing lipids, and IFABP^−/− mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP^−/−, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP^−/− mice, result in divergent downstream effects at the systemic level.     

Diversity of fatty acid-binding protein structure and function: studies with fluorescent ligands

The mammalian fatty acid-binding proteins (FABP) are localized in many distinct cell types. They bind long chain fatty acidsin vitro, however, their functions and mechanisms of actionin vivo remain unknown. The present studies have sought to understand the relationships among these proteins, and to address the possible role of FABP in cellular fatty acid traffic. A series of anthroyloxy-labeled fluorescent fatty acids have been used to examine the physicochemical properties of the fatty acid-binding sites of different members of the FABP family. The fatty acid probes have also been used to study the rate and mechanism of fatty acid transfer from different FABP types to phospholipid membranes. The results of these studies show a number of interesting and potentially important differences between FABP family members. An examination of adipocyte and heart FABP (A- and H-FABP) shows that their fatty acid-binding sites are less hydrophobic than the liver FABP (L-FABP) site, and that the bound ligand experiences less motional constraint within the A- and H-FABP binding sites than within the L-FABP binding site. In keeping with these differences in structural properties, it was found that anthroyloxy-fatty acid transfer from A- and H-FABP to membranes is markedly faster than from L-FABP. Moreover, the mechanism of fatty acid transfer was found to be similar for the highly homologous logous A- and H-FABP, whereby transfer to phospholipid membranes appears to occur via transient collisional interactions between the FABP and membranes. Transfer of fatty acids from L-FABP, in contrast, occurs via an aqueous phase diffusion mechanism. Other studies utilized fluorescent fatty acid and monoacylglycerol derivatives to compare how the two FABP which are present in high abundance in the proximal small intestine interact with the two major products of dietary triacylglycerol hydrolysis. The results showed that whereas L-FABP binds both fatty acid and monoacylglycerol derivatives, intestinal FABP (I-FABP) appears to bind fatty acid but not monoacylglycerol. In summary, studies with fluorescent ligands have demonstrated unique properties for different FABP family members. A number of these differences appear to correlate with the degree of primary sequence homology between the proteins, and suggest functional diversity within the FABP family.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - A-FABP Adipocyte FABP - I-FABP Intestinal FABP - AOffa n-(9-anthroyloxy)fatty acid - MG Monoacylglycerol - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine     

Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions

Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.     

The proximal intestinal Fatty Acid-Binding Proteins liver FABP (LFABP) and intestinal FABP (IFABP) differentially modulate whole body energy homeostasis but are not centrally involved in net dietary lipid absorption: Studies of the LFABP/IFABP double knockout mouse

Proximal intestinal enterocytes expresses both intestinal-fatty acid binding protein (IFABP; FABP2) and liver-FABP (LFABP; FABP1). These FABPs are thought to be important in the net uptake of dietary lipid from the intestinal lumen, however their specific and potentially unique functions in the enterocyte remain incompletely understood. We previously showed markedly divergent phenotypes in LFABP^?/? vs. IFABP^?/? mice fed high-fat diets, with the former becoming obese and the latter remaining lean relative to wild-type (WT) mice, supporting different functional roles for each protein. Interestingly, neither mouse model displayed increased fecal lipid concentration, raising the question of whether the presence of one FABP was sufficient to compensate for absence of the other. Here, we generated an LFABP and IFABP double knockout mouse (DKO) to determine whether simultaneous ablation would lead to fat malabsorption, and to further interrogate the individual vs. overlapping functions of these proteins. Male WT, IFABP^?/?, LFABP^?/?, and DKO mice were fed a low-fat (10 % kcal) or high-fat (45 % kcal) diet for 12 weeks. The body weights and fat mass of the DKO mice integrated those of the LFABP^?/? and IFABP^?/? single knockouts, supporting the notion that IFABP and LFABP have distinct functions in intestinal lipid assimilation that result in downstream alterations in systemic energy metabolism. Remarkably, no differences in fecal fat concentrations were found in the DKO compared to WT, revealing that the FABPs are not required for net intestinal uptake of dietary lipid.     

Interaction of enterocyte FABPs with phospholipid membranes: clues for specific physiological roles

Intestinal and liver fatty acid binding proteins (IFABP and LFABP, respectively) are cytosolic soluble proteins with the capacity to bind and transport hydrophobic ligands between different sub-cellular compartments. Their functions are still not clear but they are supposed to be involved in lipid trafficking and metabolism, cell growth, and regulation of several other processes, like cell differentiation. Here we investigated the interaction of these proteins with different models of phospholipid membrane vesicles in order to achieve further insight into their specificity within the enterocyte. A combination of biophysical and biochemical techniques allowed us to determine affinities of these proteins to membranes, the way phospholipid composition and vesicle size and curvature modulate such interaction, as well as the effect of protein binding on the integrity of the membrane structure. We demonstrate here that, besides their apparently opposite ligand transfer mechanisms, both LFABP and IFABP are able to interact with phospholipid membranes, but the factors that modulate such interactions are different for each protein, further implying different roles for IFABP and LFABP in the intracellular context. These results contribute to the proposed central role of intestinal FABPs in the lipid traffic within enterocytes as well as in the regulation of more complex cellular processes.     

Binding of 13-HODE and 15-HETE to phospholipid bilayers, albumin, and intracellular fatty acid binding proteins. implications for transmembrane and intracellular transport and for protection from lipid peroxidation

Transport and utilization of fatty acids (FA) in cells is a multistep process that includes adsorption to and movement across the plasma membrane and binding to intracellular fatty acid binding proteins (FABP) in the cytosol. We monitored the transbilayer movement of several polyunsaturated FA and oxidation products (13-hydroxy octadecadienoic acid (HODE) and 15-hydroxytetraenoic acid (HETE)) in unilamellar protein-free phospholipid vesicles containing a fluorescent pH probe. All FA diffused rapidly by the flip-flop mechanism across the model membrane, as revealed by pH changes inside the vesicle. This result suggests that FA oxidation products generated in the cell could cross the plasma or nuclear membrane spontaneously without a membrane transporter. To illuminate features of extra- and intracellular transport, the partitioning of unsaturated FA and oxidized FA between phospholipid vesicles and albumin or FABP was studied by the pyranin assay. These experiments showed that all polyunsaturated FA and oxidized FA (13-HODE and 15-HETE) desorbed rapidly from the phospholipid bilayer to bind to bovine serum albumin, which showed a slight preference for the unsaturated FA over the oxidized FA. FABP rapidly bound FA in the presence of phospholipid bilayers, with a preference of 13-HODE over the unsaturated FA and with a specificity depending on the type of FABP. Liver FABP was significantly more effective than intestinal FABP in binding 13-HODE in the presence of vesicles. The more effective binding of the FA metabolite, 13-HODE, than its precursor 18:2 by FABP may help protect cellular membranes from potential damage by monohydroxy fatty acids and may contribute a pathway for entry of 13-HODE into the nucleus.     

FABP1 knockdown in human enterocytes impairs proliferation and alters lipid metabolism

Fatty Acid-Binding Proteins (FABPs) are abundant intracellular proteins that bind long chain fatty acids (FA) and have been related with inmunometabolic diseases. Intestinal epithelial cells express two isoforms of FABPs: liver FABP (LFABP or FABP1) and intestinal FABP (IFABP or FABP2). They are thought to be associated with intracellular dietary lipid transport and trafficking towards diverse cell fates. But still their specific functions are not well understood.To study FABP1's functions, we generated an FABP1 knockdown model in Caco-2 cell line by stable antisense cDNA transfection (FABP1as). In these cells FABP1 expression was reduced up to 87%. No compensatory increase in FABP2 was observed, strengthening the idea of differential functions of both isoforms. In differentiated FABP1as cells, apical administration of oleate showed a decrease in its initial uptake rate and in long term incorporation compared with control cells. FABP1 depletion also reduced basolateral oleate secretion. The secreted oleate distribution showed an increase in FA/triacylglyceride ratio compared to control cells, probably due to FABP1's role in chylomicron assembly. Interestingly, FABP1as cells exhibited a dramatic decrease in proliferation rate. A reduction in oleate uptake as well as a decrease in its incorporation into the phospholipid fraction was observed in proliferating cells.Overall, our studies indicate that FABP1 is essential for proper lipid metabolism in differentiated enterocytes, particularly concerning fatty acids uptake and its basolateral secretion. Moreover, we show that FABP1 is required for enterocyte proliferation, suggesting that it may contribute to intestinal homeostasis.     

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.     

Mechanism of free fatty acid transfer from rat heart fatty acid-binding protein to phospholipid membranes. Evidence for a collisional process.

Fatty acid binding proteins (FABP) are a family of low molecular weight proteins found in many tissues that actively utilize free fatty acids (ffa). FABP would be expected to have a particularly important role in the heart, where over 80% of energy requirements are derived from oxidation of long chain fatty acids. The precise physiological function of heart FABP (H-FABP) has not been definitively identified, although it is thought to play a role in intracellular ffa transport. To examine the possible role of H-FABP in cardiac myocyte transfer of ffa, we examined the transfer of fluorescent anthroyloxy ffa (AOffa) from H-FABP to model phospholipid membranes, using a resonance energy transfer assay. In contrast to previous observations of ffa transfer from liver FABP and from membranes, transfer from H-FABP to membranes appears to occur by a different mechanism. AO-palmitate (16:0) transfer was 1.5-fold slower than AO-stearate (18:0) transfer, and mono-unsaturation did not affect the transfer rate. The AOffa transfer rate from H-FABP increased with increasing ionic strength and decreased slightly between pH 7 and 9. These results suggest that the rate of ffa transfer from H-FABP to membranes is independent of the ffa aqueous solubility. Thermodynamic analysis showed that the free energy of activation for the ffa transfer process arises primarily from an enthalpic component, with only a small entropic contribution, again suggesting the lack of an aqueous phase route of ffa delivery. Finally, the ffa transfer rate was found to be directly dependent on the concentration of acceptor membranes. These data therefore suggest that transfer of AOffa from H-FABP to membranes may occur via collisional interactions between the protein and membranes.     

Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms

Intestinal enterocytes contain high concentrations of two cytosolic fatty acid-binding proteins (FABP), liver FABP (L-FABP) and intestinal FABP (I-FABP), which are hypothesized to play a role in cellular fatty acid trafficking. The mechanism(s) by which fatty acids move from membranes to each of these proteins is not known. Here we demonstrate that fluorescent anthroyloxy fatty acid analogues (AOFA) are transferred from phospholipid vesicles to L-FABP versus I-FABP by different mechanisms. For L-FABP a diffusion-mediated transfer process is demonstrated. The AOFA transfer rate from phosphatidylcholine-containing vesicles (POPC) to L-FABP is similar to that observed with another diffusional process, namely inter-membrane AOFA transfer. Furthermore, the AOFA transfer rate was modulated by buffer ionic strength and AOFA solubility, while the transfer rate remained relatively unchanged by the presence of anionic phospholipids in vesicles. In contrast, the data for I-FABP suggest that a transient collisional interaction of I-FABP with the phospholipid membrane occurs during AOFA extraction from the vesicles by the protein. In particular, the presence of the anionic phospholipid cardiolipin in donor vesicles increased the rate of AOFA transfer to I-FABP by 15-fold compared with transfer to POPC vesicles. The effects of ionic strength on transfer suggest that the interaction of I-FABP with cardiolipin-containing vesicles is likely to contain an electrostatic component. Finally, based on the regulation of AOFA transfer to I-FABP compared with transfer from I-FABP, it is hypothesized that apo- and holo-I-FABPs adopt conformations which may differentially promote I-FABP-membrane interactions.In summary, the results suggest that I-FABP, but not L-FABP, can directly extract fatty acids from membranes, supporting the concept that I-FABP may increase the cytosolic flux of fatty acids via intermembrane transfer.     

Conformational exchange of fatty acid binding protein induced by protein-nanodisc interactions

Fatty acid binding proteins (FABPs) can facilitate the transfer of long-chain fatty acids between intracellular membranes across considerable distances. The transfer process involves fatty acids, their donor membrane and acceptor membrane, and FABPs, implying that potential protein-membrane interactions exist. Despite intensive studies on FABP-membrane interactions, the interaction mode remains elusive, and the protein-membrane association and dissociation rates are inconsistent. In this study, we used nanodiscs (NDs) as mimetic membranes to investigate FABP-membrane interactions. Our NMR experiments showed that human intestinal FABP interacts weakly with both negatively charged and neutral membranes, but it prefers the negatively charged one. Through simultaneous analysis of NMR relaxation in the rotating-frame (R_1ρ), relaxation dispersion, chemical exchange saturation transfer, and dark-state exchange saturation transfer data, we estimated the affinity of the protein to negatively charged NDs, the dissociation rate, and apparent association rate. We further showed that the protein in the ND-bound state adopts a conformation different from the native structure and the second helix is very likely involved in interactions with NDs. We also found a membrane-induced FABP conformational state that exists only in the presence of NDs. This state is native-like, different from other conformational states in structure, unbound to NDs, and in dynamic equilibrium with the ND-bound state.