A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins

Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family.     

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP^−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

Fatty acid induced remodeling within the human liver fatty acid-binding protein

We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP.     

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.     

Temperature-induced conformational switch in intestinal fatty acid binding protein (IFABP) revealing an alternative mode for ligand binding

IFABP is a small beta-barrel protein with a short helix-turn-helix motif near the N-terminus that is thought to participate in the regulation of the uptake and delivery of fatty acids. In a previous work, we detected by near UV circular dichroism a reversible conformational transition of this protein occurring between 35 and 50 degrees C in the absence of fatty acids. The addition of the natural ligand oleic acid prevents this phenomenon. In both cases, the overall structure of the beta-barrel is maintained. This thermal transition is also detected by the fluorescent probe bis-anilino naphthalene sulfonic acid (bisANS) but not by its monomer ANS. In the present work, we studied in detail the interaction of each compound with IFABP as a function of temperature and in the absence or in the presence of oleic acid. A contrasting behavior was observed for these probes: (i) IFABP is able to bind two molecules of bisANS but only one molecule of ANS and (ii) oleic acid can fully displace ANS but only partially bisANS. Three independent lines of evidence, namely, fluorescence spectroscopy, circular dichroism, and limited proteolysis, indicate that there is an equilibrium among different conformations of IFABP, which differ in the extent of flexibility of the helical domain. This equilibrium can be shifted by raising temperature. bisANS is able to probe a population of IFABP in an altered state, which is more susceptible to cleavage by clostripain as compared to the apo-form, whereas the conformation of IFABP bound to oleic acid is characteristically more ordered. These results highlight the idea of an enhanced flexibility exhibited by IFABP that bears importance on its transport function, supporting the role of a dynamic entry portal region for the fatty acid ligand.     

Molecular Dynamics Simulation of Ligand Dissociation from Liver Fatty Acid Binding Protein

The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized “portal region” could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques.     

Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes

Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.     

Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles

The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --> isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions.     

Expression of rat intestinal fatty acid-binding protein in Escherichia coli. Purification and comparison of ligand binding characteristics with that of Escherichia coli-derived rat liver fatty acid-binding protein

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.     

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.     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations

Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a beta-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.     

Protocols and pitfalls in obtaining fatty acid-binding proteins for biophysical studies of ligand-protein and protein-protein interactions

Adipocyte fatty acid-binding protein (AFABP: FABP4) is a member of the intracellular lipid-binding protein family that is thought to target long-chain fatty acids to nuclear receptors such as peroxisome proliferator-activated receptor gamma (PPARγ), which in turn plays roles in insulin resistance and obesity. A molecular understanding of AFABP function requires robust isolation of the protein in liganded and free forms as well as characterization of its oligomerization state(s) under physiological conditions. We report development of a protocol to optimize the production of members of this protein family in pure form, including removal of their bound lipids by mixing with hydrophobically functionalized hydroxypropyl dextran beads and validation by two-dimensional NMR spectroscopy. The formation of self-associated or covalently bonded protein dimers was evaluated critically using gel filtration chromatography, revealing conditions that promote or prevent formation of disulfide-linked homodimers. The resulting scheme provides a solid foundation for future investigations of AFABP interactions with key ligand and protein partners involved in lipid metabolism.     

Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 1. Adenosine conformations by proton two-dimensional transferred nuclear Overhauser effect spectroscopy

Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP), and of an ATP analogue, adenylyl imidodiphosphate (AMPPNP), bound to Escherichia coliadenylate kinase (AKe) in the complexes of AKe.Mg(II)ATP, AKe.AMP.Mg(II)GDP, AKe. AMPPNP, and AKe.Mg(II)AMPPNP were determined by transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements and molecular dynamics simulations. The glycosidic torsion angles, chi, deduced for the adenine nucleotides in these complexes are 51 degrees, 37 degrees, 49 degrees, and 47 degrees, respectively, with an experimental error of about +/-5 degrees. These values are in general agreement with those previously measured for other ATP-utilizing enzymes, suggesting a possible common motif for adenosine recognition and binding. The pseudorotational phase angle, P, of the sugar puckers for the bound nucleotides varied between 50 degrees and 103 degrees. These solution-state conformations are significantly different from those in published data from X-ray crystallography. A computation of the ligand NOEs, made by using the program CORCEMA [Moseley, H. N. B., Curto, E. V., and Krishna, N. R. (1995) J. Magn. Reson. B108, 243-261] with the protein protons in the vicinity of nucleotide included, on the basis of the X-ray structure of the AKe.AMP.AMPPNP complex [Berry, M. B., Meador, B., Bilderback, T., Liang, P., Glaser, M., and Philips, G. N. , Jr. (1994) Proteins: Struct., Funct., Genet. 19, 183-198], showed that polarization transfer to the protein protons does not produce significant errors in the structures determined by considering the ligand NOEs alone.     

Polyene fatty acid interactions with recombinant intestinal and liver fatty acid-binding proteins. Spectroscopic studies

Binding and proximity relationships of fatty acids with recombinant rat liver fatty acid-binding protein (L-FABP) and intestinal fatty acid-binding protein (I-FABP) were studied with absorption and fluorescence spectroscopy. Protein aromatic amino acids were examined in the absence and presence of bound fatty acid. Second derivative absorbance spectroscopy of the apo- and holoproteins suggested that fatty acid binding altered the conformation of L-FABP, but not of I-FABP. Fatty acid binding also blocked the accessibility of L-FABP tyrosine and I-FABP tryptophan to Stern-Volmer quenching by acrylamide, indicating that these amino acids were present in the fatty acid-binding pocket. Forster energy transfer from I-FABP tryptophan to bound cis-parinaric acid resulted in quenching of tryptophan lifetime and appearance of sensitized lifetime of bound cis-parinaric acid. The calculated donor-acceptor distances were 16.9 +/- 0.6 and 19.2 +/- 0.3 A for I-FABP and L-FABP, respectively. Absorbance spectral shifts and ratios of fluorescence excitation maxima indicated that the parinaric acid microenvironment in the fatty acid-binding site of I-FABP was much less polar than that of L-FABP. Parinaric acids displayed similar rotational correlation time and limiting anisotropy when bound to I-FABP and to L-FABP. These results are consistent with a close proximity of bound fatty acids to the tyrosine and tryptophan residues and with immobilization of the polyene fatty acids in the fatty acid-binding site(s) of L-FABP and I-FABP. The two proteins differ in that only L-FABP has two fatty acid-binding sites and appears to undergo significant conformational change upon fatty acid binding.     

Tryptophan insertion mutagenesis of liver fatty acid-binding protein: L28W mutant provides important insights into ligand binding and physiological function

Liver fatty acid-binding protein (FABP) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, and bile acids. Previously we prepared charge reversal mutants and demonstrated the importance of lysine residues within the portal region in ligand and membrane binding. We have now prepared several tryptophan-containing mutants within the portal region, and one tryptophan at position 28 (L28W) has proved remarkably effective as an intrinsic probe to further study ligand binding. The fluorescence of the L28W mutant was very sensitive to fatty acid and bile acid binding where a large (up to 4-fold) fluorescence enhancement was obtained. In contrast, the binding of oleoyl CoA reduced tryptophan fluorescence. Positive cooperativity for fatty acid binding was observed while detailed information on the orientation of binding of bile acid derivatives was obtained. The ability of bound oleoyl CoA to reduce the fluorescence of L28W provided an opportunity to demonstrate that fatty acyl CoAs can compete with fatty acids for binding to liver FABP under physiological conditions, further highlighting the role of fatty acyl CoAs in modulating FABP function in the cell.     

The pH-dependent unfolding mechanism of P2 myelin protein: an experimental and computational study

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands beta-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.     

Characterization of a fatty acid-binding protein from rat heart

A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed.     

Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings

Residual dipolar couplings for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This approach is used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin. Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (+/-4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide.