Crystallographic studies of protein denaturation and renaturation. 1. Effects of denaturants on volume and X-ray pattern of cross-linked triclinic lysozyme crystals

Triclinic crystals of hen egg-white lysozyme cross-linked with glutaraldehyde have been treated with various denaturants and found to be susceptible to x-ray structure analysis even after major conformational changes in the protein. Cross-linked crystals were isomorphous with the native form, and electron density difference maps indicated the locations of intermolecular corss-links, but showed no appreciable differences in the protein conformation. Soaking of the cross-linked crystals in danaturant solutions of increasing concentrations caused corresponding increases in crystal volume and decreases in minimum observable x-ray spacings. These changes proved partly reversible on diluting the solutions, and measurements of crystal volume and minimums x-ray spacing were used to follow denaturation and renaturation as a function of concentration for several denaturants. Some of these, including bromoethanol and sodium dodecyl sulfate, had little effect on the crystals below critical concentrations at which there was a sharp volume increase and loss of x-ray pattern, which could, however, be regenerated to about 3.2-A resolution. Others, including KCNS and urea, caused more gradual changes, but with a smaller degree of recovery. It is suggested that at least two different denaturation mechanisms are involved with detergent-like reagents disrupting the hydrophobic interactions joining the two wings of the lysozyme molecule and hydrophilic denaturants interacting primarily with polar groups on the molecular surface.     

Fluorine-19 NMR studies on the acid state of the intestinal fatty acid binding protein

The intestinal fatty acid binding protein (IFABP) is composed of two beta-sheets with a large hydrophobic cavity into which ligands bind. After eight 4-(19)F-phenylalanines were incorporated into the protein, the acid state of both apo- and holo-IFABP (at pH 2.8 and 2.3) was characterized by means of (1)H NMR diffusion measurements, circular dichroism, and (19)F NMR. Diffusion measurements show a moderately increased hydrodynamic radius while near- and far-UV CD measurements suggest that the acid state has substantial secondary structure as well as persistent tertiary interactions. At pH 2.8, these tertiary interactions have been further characterized by (19)F NMR and show an NOE cross-peak between residues that are located on different beta-strands. Side chain conformational heterogeneity on the millisecond time scale was captured by phase-sensitive (19)F-(19)F NOESY. At pH 2.3, native NMR peaks are mostly gone, but the protein can still bind fatty acid to form the holoprotein. An exchange cross-peak of one phenylalanine in the holoprotein is attributed to increased motional freedom of the fatty acid backbone caused by the slight opening of the binding pocket at pH 2.8. In the acid environment Phe128 and Phe17 show dramatic line broadening and chemical shift changes, reflecting greater degrees of motion around these residues. We propose that there is a separation of specific regions of the protein that gives rise to the larger radius of hydration. Temperature and urea unfolding studies indicate that persistent hydrophobic clusters are nativelike and may account for the ability of ligand to bind and induce nativelike structure, even at pH 2.3.     

Steady-state and time-resolved fluorescence studies of the intestinal fatty acid binding protein

The intestinal fatty acid binding protein contains two tryptophan residues (Trp6 and Trp82) both of which have been shown by X-ray and NMR methods to be buried in hydrophobic clusters. By using a combination of steady-state and time-resolved fluorescence experiments, we have deconvoluted the lifetime weighted contribution of each of the tryptophans to the steady-state fluorescence quantum yield. While Trp82 has been implicated in an intermediate that appears at relatively high denaturant concentrations, the variation of the lifetime weighted contribution of Trp6 with urea or guanidium hydrochloride shows formation of an intermediate state at low concentrations of the denaturant before the actual unfolding starts. Trp82 did not show similar behavior. Fluorescence quenching experiments by acrylamide show that while Trp6 in the native protein is less solvent-exposed, its accessibility is increased significantly at low urea concentration indicating that the early intermediate state is partially unfolded. Time-resolved anisotropy experiments indicate that the volume of the partially unfolded intermediates is larger than the native protein and lead to the speculation that the last step of the protein folding might be the removal of solvent molecules from the protein.     

Crystallization of rat intestinal fatty acid binding protein. Preliminary X-ray data obtained from protein expressed in Escherichia coli

Rat intestinal fatty acid binding protein has been expressed in Escherichia coli, purified with bound long chain fatty acids and crystals grown from solutions of polyethylene glycol 4000. The crystals are monoclinic, space group P2(1), a = 3638 A, b = 57.2 A, c = 31.9 A, and beta = 113.9 degrees. Each unit cell contains two monomers of this 132-residue, 15.1-kDa polypeptide. The crystals are remarkably resistant to x-ray damage. X-ray diffraction data have been observed to 2.0 A resolution. Platinum chloride was used to generate a potential isomorphous heavy atom derivative.     

Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.     

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.     

Self-association of the cardiac fatty acid binding protein. Influence on membrane-bound, fatty acid dependent enzymes

The present study on the fatty acid binding protein, purified from pig heart and studied by three independent techniques (electron spin resonance, circular dichroism, and polyacrylamide gel electrophoresis), suggests that the protein self-aggregates and exists in at least four distinct molecular species. This plurality is demonstrated by the presence of four bands after electrophoretic migration at pH 7.2 and by three transitions of molar ellipticity theta 225 that depend on protein concentration. A mathematical model is formulated to simulate the three transitions and to calculate the concentrations of the four species. The multistates manifest themselves in a complex binding capacity for fatty acid, with two sigmoidal components in the binding curve. A general equation for the curve is formulated, and the characteristic constants are evaluated by a nonlinear least-squares fit. The experimental results and their interpretation in quantitative terms lead to a theoretical evaluation of the importance of this new property of self-aggregation of the protein on the activity of membrane-bound model enzymes which are fatty acid or acyl coenzyme A dependent.     

Liver fatty acid binding protein expression enhances branched-chain fatty acid metabolism

Although liver fatty acid binding protein (L-FABP) is known to enhance uptake and esterification of straight-chain fatty acids such as palmitic acid and oleic acid, its effects on oxidation and further metabolism of branched-chain fatty acids such as phytanic acid are not completely understood. The present data demonstrate for the first time that expression of L-FABP enhanced initial rate and average maximal oxidation of [2,3-3H] phytanic acid 3.5- and 1.5-fold, respectively. This enhancement was not due to increased [2,3-3H] phytanic acid uptake, which was only slightly stimulated (20%) in L-FABP expressing cells after 30 min. Similarly, L-FABP also enhanced the average maximal oxidation of [9,10-3H] palmitic acid 2.2-fold after incubation for 30 min. However, the stimulation of L-FABP on palmitic acid oxidation nearly paralleled its 3.3-fold enhancement of uptake. To determine effects of metabolism on fatty acid uptake, a non-metabolizable fluorescent saturated fatty acid, BODIPY-C16, was examined by laser scanning confocal microscopy (LSCM). L-FABP expression enhanced uptake of BODIPY-C16 1.7-fold demonstrating that L-FABP enhanced saturated fatty acid uptake independent of metabolism. Finally, L-FABP expression did not significantly alter [2,3-3H] phytanic acid esterification, but increased [9,10-3H] palmitic acid esterification 4.5-fold, primarily into phospholipids (3.7-fold) and neutral lipids (9-fold). In summary, L-FABP expression enhanced branched-chain phytanic acid oxidation much more than either its uptake or esterification. These data demonstrate a potential role for L-FABP in the peroxisomal oxidation of branched-chain fatty acids in intact cells.     

Binding properties of Echinococcus granulosus fatty acid binding protein.

EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.     

Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes.     

Intestinal fatty acid binding protein may favor differential apical fatty acid binding in the intestine

The intestinal mucosa metabolizes fatty acids differently when presented to the lumenal or basolateral membrane. Expression of both liver and intestinal fatty acid binding proteins (L- and I-FABPs) uniquely in the enterocyte offers a possible explanation of this phenomenon. An organ explant system was used to analyze the relative binding of fatty acids to each protein. More fatty acid was bound to L-FABP than to I-FABPs (28% vs. 6% of cytosolic radioactivity), no matter on which side the fatty acid was added. However, a 2-3-fold increase in fatty acid binding to the intestinal paralog was noted after apical addition of palmitic or oleic acid in mucosa from chow fed rats. When oleic acid was added apically, a 1.4-fold increase in binding to I-FABP was observed in mucosa derived from chronically fat fed rats, consistent with the previously observed 50% increase in the content of that protein. Immunocytochemical localization of both FABPs in vivo demonstrated an apical cytoplasmic localization in the fasting state, and redistribution to the entire cytoplasm after fat feeding. These data are consistent with the hypothesis that I-FABP may contribute to the metabolic compartmentalization of apically presented fatty acids in the intestine.     

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.     

Hierarchical folding of intestinal fatty acid binding protein

Intestinal fatty acid binding protein (IFABP) is a member of the lipid binding protein family, members of which have a clam shell type of motif formed by two five-stranded beta-sheets. Understanding the folding mechanism of these proteins has been hindered by the presence of an unresolved burst phase. By initiating the reaction with a sub-millisecond mixer and following its progression by Trp fluorescence, we discovered three distinct phases in the folding reaction of the W6Y mutant of IFABP from which we postulate the following sequence of events. The first phase (k(1) > 10 000 s(-1)) involves collapse of the polypeptide chain around a hydrophobic core. During the second phase (k(2) approximately 1500 s(-1)), beta-strands B-G, mostly located on the top half of the clam shell structure, propagate from this hydrophobic core. It is followed by the final phase (k(3) approximately 5 s(-1)) involving the formation of the last three beta-strands on the bottom half of the clam shell and the establishment of the native hydrogen bonding network throughout the protein molecule.     

Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an "open-cap" conformation or a "swivel-back" mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics.     

Intestinal fatty acid binding protein: the folding mechanism as determined by NMR studies

The intestinal fatty acid binding protein is composed of two beta-sheets surrounding a large interior cavity. There is a small helical domain associated with the portal for entry of the ligand into the cavity. Denaturation of the protein has been monitored in a residue-specific manner by collecting a series of two-dimensional (1)H-(15)N heteronuclear single-quantum coherence (HSQC) NMR spectra from 0 to 6.5 M urea under equilibrium conditions. In addition, rates for hydrogen-deuterium exchange have been measured as a function of denaturant concentration. Residual, native-like structure persists around hydrophobic clusters at very high urea concentrations. This residual structure (reflecting only about 2-7% persistence of native-like structure) involves the turns between beta-strands and between the two short helices. If this persistence is assumed to reflect transient native-like structure in these regions of the polypeptide chain, these sites may serve as nucleation sites for folding. The data obtained at different urea concentrations are then analyzed on the basis of peak intensities relative to the intensities in the absence of urea reflecting the extent of secondary structure formation. At urea concentrations somewhat below 6.5 M, specific hydrophobic residues in the C-terminal beta-sheet interact and two strands, the D and E strands in the N-terminal beta-sheet, are stabilized. These latter strands surround one of the turns showing residual structure. With decreasing urea concentrations, the remaining strands are stabilized in a specific order. The early strand stabilization appears to trigger the formation of the remainder of the C-terminal beta-sheet. At low urea concentrations, hydrogen bonds are formed. A pathway is proposed on the basis of the data describing the early, intermediate, and late folding steps for this almost all beta-sheet protein. The data also show that there are regions of the protein which appear to act in a concerted manner at intermediate steps in refolding.     

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.     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

Spectroscopic investigations on the binding site of bovine hepatic fatty acid binding protein. Evidence for the existence of a single binding site for two fatty acid molecules

The hydrophobic region of the binding site of a bovine fatty acid binding protein (pI 7.0-FABP) has been characterized using fluorescence and circular dichroism (CD) spectroscopy. Blue-shifts of fluorescence emission maxima and increased lifetimes of naphthylamine dyes, anthroyloxy-fatty acids, pyrene nonanoic acid and trans-parinaric acid indicated a hydrophobic interaction with FABP. The fluorescence quenching of various anthroyloxy-fatty acids by iodide and acrylamide showed lower accessibility to the fluorophore linked to the carbon adjacent to the carbonyl group and towards the methyl end of the fatty acid. Binding stoichiometries were different for fatty acids and their bulky fluorescent analogues. trans-Parinaric acid when bound to FABP showed a complex induced CD-spectrum, which is explained by a close proximity of two ligands in the same binding site. Fluorescent derivatives of phosphatidylcholine with trans-parinaric acid and cholesteryl trans-parinarate did not bind to FABP. Thus, the binding site appears to be constructed for high affinity binding of long chain fatty acids.     

Effect of branched-chain fatty acid on lipid dynamics in mice lacking liver fatty acid binding protein gene

Although a role for liver fatty acid protein (L-FABP) in the metabolism of branched-chain fatty acids has been suggested based on data obtained with cultured cells, the physiological significance of this observation remains to be demonstrated. To address this issue, the lipid phenotype and metabolism of phytanic acid, a branched-chain fatty acid, were determined in L-FABP gene-ablated mice fed a diet with and without 1% phytol (a metabolic precursor to phytanic acid). In response to dietary phytol, L-FABP gene ablation exhibited a gender-dependent lipid phenotype. Livers of phytol-fed female L-FABP–/– mice had significantly more fatty lipid droplets than male L-FABP–/– mice, whereas in phytol-fed wild-type L-FABP+/+ mice differences between males and females were not significant. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice. These results were reflected in the lipid profile, where hepatic levels of triacylglycerides in phytol-fed female L-FABP–/– mice were significantly higher than in male L-FABP–/– mice. Furthermore, livers of phytol-fed female L-FABP–/– mice exhibited more necrosis than their male counterparts, consistent with the accumulation of higher levels of phytol metabolites (phytanic acid, pristanic acid) in liver and serum, in addition to increased hepatic levels of sterol carrier protein (SCP)-x, the only known peroxisomal enzyme specifically required for branched-chain fatty acid oxidation. In summary, L-FABP gene ablation exerted a significant role, especially in female mice, in branched-chain fatty acid metabolism. These effects were only partially compensated by concomitant upregulation of SCP-x in response to L-FABP gene ablation and dietary phytol. gene targeting; phytanic acid