Rat intestinal fatty acid binding protein. A model system for analyzing the forces that can bind fatty acids to proteins.
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Animals↗
Biomedical subjects
J C Sacchettini
Publications and source records attributed to J C Sacchettini.
The 2.0-A resolution structure of soybean beta-amylase complexed with alpha-cyclodextrin.
New crystallographic findings are presented which offer a deeper understanding of the structure and functioning of beta-amylase, the first known exo-type starch-hydrolyzing enzyme. A refined three-dimensional structure of soybean beta-amylase, complexed with the inhibitor alpha-cyclodextrin, has been determined at 2.0-A resolution with a conventional R-value of 17.5%. The model contains 491 amino acid residues, 319 water molecules, 1 sulfate ion, and 1 alpha-cyclodextrin molecule. The protein consists of a core with an (alpha/beta)8 supersecondary structure, plus a smaller globular region formed by long loops (L3, L4, and L5) extending from beta-strands beta 3, beta 4, and beta 5. Between the two regions is a cleft that opens into a pocket whose floor contains the postulated catalytic center near the carboxyl group of Glu 186. The annular alpha-cyclodextrin binds in (and partly projects from) the cleft with its glucosyl O-2/O-3 face abutting the (alpha/beta)8 side and with its alpha-D(1 --> 4) glucosidic linkage progression running clockwise as viewed from that side. The ligand does not bind deeply enough to interact with the carboxyl group of Glu 186. Rather, it occupies most of the cleft entrance, strongly suggesting that alpha-cyclodextrin inhibits catalysis by blocking substrate access to the more deeply located reaction center. Of the various alpha-cyclodextrin interactions with protein residues in loops L4, L5, L6, and L7, most notable is the shallow inclusion complex formed with Leu 383 (in L7, on the core side of the cleft) through contacts of its methyl groups with the C-3 atoms of four of the ligand's D-glucopyranosyl residues. All six residues of the bound alpha-cyclodextrin are of 4C1 conformation and are joined by alpha-1,4 linkages with similar torsional angles to form a nearly symmetrical torus as reported for crystalline inclusion complexes with alpha-cyclodextrin. We envision a significant role for the methyl groups of Leu 383 at the cleft entrance with respect to the productive binding of the outer chains of starch.
Amino Acid Sequence↗
Primary structure and crystallization of orotate phosphoribosyltransferase from Salmonella typhimurium.
Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes phosphoribosyl group transfer between alpha-D-5-phosphoribosyl-1-pyrophosphate and orotate to form orotidine-5'-monophosphate and pyrophosphate, the nucleotide-forming step in pyrimidine biosynthesis. It is one of ten PRTases that perform vital roles in de novo and salvage pathways for purine, pyrimidine and pyridine nucleotides. Although the PRTases are important drug targets, they are poorly understood mechanistically, and no three-dimensional structures exist. Here, we report the complete sequence of the Salmonella typhimurium pyrE gene and the deduced sequence of the OPRTase gene product. OPRTase forms tetragonal crystals from polyethylene glycol solutions; these crystals diffract to better than 2 A resolution, and are stable to radiation damage. The space group is P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 48.5 A, c = 210.5 A, and alpha = beta = gamma = 90 degrees. A crystalline form of the selenomethionine derivative of the protein is also reported.
Amino Acid Sequence↗
Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-A resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate.
The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg106, the nitrogen of the indole group of Trp82, and the side chain of Gln115. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr70 and Tyr117 envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The omega-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe55 allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I-FABP:oleate with a recently refined 1.19-A model of apoI-FABP and an earlier 2.0-A model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between beta strands C and D, and the ring of Phe55 together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe55 which shows discrete disorder in the apoprotein functions as an omega-terminal "sensing device": moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr70 and Tyr117 which also show discrete disorder in the apoprotein structure due to rotation around their C alpha-C beta bonds, are stabilized in a single, well ordered position in the holoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Three-dimensional structure of recombinant human muscle fatty acid-binding protein.
The three-dimensional structure of recombinant human muscle fatty acid-binding protein with a bound fatty acid has been solved and refined with x-ray diffraction data to 2.1 A resolution. The refined model has a crystallographic R factor of 19.5% for data between 9.0 and 2.1 A (7243 unique reflections) and root-mean-square deviations in bond length and bond angle of 0.013 A and 2.7 degrees. The protein contains 10 antiparallel beta-strands and two short alpha-helices which are arranged into two approximately orthogonal beta-sheets. Difference electron density maps and a multiple isomorphous derivative electron density map showed the presence of a single bound molecule of a long chain fatty acid within the interior core of the protein. The hydrocarbon tail of the fatty acid was found to be in a "U-shaped" conformation. Seven ordered water molecules were also identified within the interior of the protein in a pocket on the pseudo-si face of the fatty acid's bent hydrocarbon tail. The methylene tail of the fatty acid forms van der Waals interactions with atoms from 13 residues and three ordered waters. The carboxylate of the fatty acid is located in the interior of the protein where it forms hydrogen bonds with the side chains of Tyr128 and Arg126 and two ordered water molecules. A comparison of the three-dimensional structure of human muscle fatty acid-binding protein and rat intestinal fatty acid-binding protein shows strong similarity. Both proteins bind a single fatty acid within their interior cores, but the bound fatty acids are very different in their conformations and interactions. These findings suggest that the intestinal and muscle fatty acid-binding proteins have evolved distinct binding sites in order to satisfy different requirements within the tissues where they are expressed.
Amino Acid Sequence↗
Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition.
To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain atoms and by contacts of its side chains with the H-2Kb molecule. The amino-terminal nitrogen atom of the peptide forms a hydrogen bond with the hydroxyl group of Tyr-171 of H-2Kb at one end of the groove, while the carboxyl-terminal oxygen forms a hydrogen bond with the hydroxyl group of Tyr-84 at the other end. Since the amino acids at both ends are conserved among human and mouse MHC molecules, this anchoring of each end of the peptide appears to be a general feature of peptide-MHC class I molecule binding and imposes restrictions on its length. The side chains of residues Tyr-3, Tyr-5, and Leu-8 of the VSV octapeptide fit into the interior of the H-2Kb molecule with no appreciable surface exposure, a finding in support of previous biological studies that showed the importance of these residues for binding. Thus, the basis for binding of specific peptide sequences to the MHC class I molecule is the steric restriction imposed on the peptide side chains by the architecture of the floor and sides of the groove. The side chains of Arg-1, Val-4, and Gln-6 and the main-chain of Gly-7 of the octapeptide are exposed on the surface of the complex, thus confirming their availability for T-cell receptor contact, as previously demonstrated by T-cell recognition experiments.
Amino Acid Sequence↗
Refinement of the structure of recombinant rat intestinal fatty acid-binding apoprotein at 1.2-A resolution.
The three-dimensional structure of the 131-residue rat intestinal fatty acid-binding protein, without bound ligand (apoI-FABP), has been refined with x-ray diffraction data to a nominal resolution of 1.19 A. The final model has a conventional crystallographic R-factor of 16.9% for 34,290 unique reflections [a root mean square (r.m.s.) deviation for bond length of 0.012 A and a r.m.s. deviation of 2.368 degrees for bond angles]. Ninety-two residues are present as components of the protein's 10 anti-parallel beta-strands while 14 residues are part of its two short alpha-helices. The beta-strands and alpha-helices are organized into two nearly orthogonal beta-sheets. Particular attention has been placed in defining solvent structure and the structures of discretely disordered groups in this protein. Two hundred thirty-seven solvent molecules have been identified; 24 are located within apoI-FABP. The refined model includes alternate conformers for 228 protein atoms (109 main-chain, 119 side-chain) and 63 solvent molecules. We have found several aromatic side-chains with multiple conformations located near, or in, the protein's ligand binding site. This observation, along with the fact that these side-chains have a temperature factor that is relatively higher than that of other aromatic residues, suggests that they may be involved in the process of noncovalent binding of fatty acid. The absence of a true hydrophobic core in I-FABP suggests that its structural integrity may be maintained primarily by a hydrogen bonding network involving protein and solvent atoms.
Amino Acid Sequence↗
Developmental and structural studies of an intracellular lipid binding protein expressed in the ileal epithelium.
Enterocytes located in the pig distal small intestine (ileum) contain a cytosolic protein that is homologous to two proteins that are also synthesized in these cells: intestinal and "liver" fatty acid-binding proteins (I- and L-FABPc, respectively). To begin to investigate the functional interrelationships of these three proteins, we compared their patterns of tissue-specific expression and developmental regulation in the mouse. Blot hybridization analyses of RNA prepared from 12 adult tissues revealed that this mRNA was confined to the small intestine. Unlike I- and L-FABPc mRNA, which are most abundant in the proximal jejunum, this mRNA is most abundant in the ileum. While I- and L-FABPc gene transcription commence in late fetal life coincident with initial cytodifferentiation of the mouse gut epithelium, the ileal gene is activated later, at the suckling/weaning transition (postnatal day 12). The ileal location and developmental pattern of expression suggested that this protein may play a role in the intracellular transport of bile salts in the ileal epithelium. To test this hypothesis, we expressed the porcine ileal peptide (PIP) in Escherichia coli, purified it to apparent homogeneity, and analyzed its binding properties for bile acids and fatty acids using 13C NMR spectroscopy. Like I-FABPc, PIP binds palmitate and oleate with a 1:1 molar stoichiometry. However, unlike I-FABPc PIP binds chenodeoxycholate. In addition, the presence of chenodeoxycholate blocks fatty acid binding to PIP, but not to I-FABPc. E. coli-derived PIP was subsequently crystallized with and without chenodeoxycholic acid. All crystals are orthorhombic in the P2(1)2(1)2(1) space group. The unit cell dimensions are a = 36.15 A, b = 50.13 A, and c = 67.18 A.
Aging↗
Gastrotropin: not an enterooxyntin but a member of a family of cytoplasmic hydrophobic ligand binding proteins.
Gastrotropin, a peptide initially isolated from porcine intestinal extracts, has been proposed to be an enterooxyntin. We have isolated a full length gastrotropin cDNA from a hog small intestinal lambda gt11 library. No evidence of co-translational translocation or processing of the encoded 127-residue protein could be demonstrated using an in vitro transcription/translation/microsomal processing assay. Comparative sequence analyses indicate that gastrotropin is a new member of the family of cytoplasmic hydrophobic ligand proteins. Analysis of the distribution of gastrotropin in nine adult Sprague-Dawley rat tissues revealed that the gene is expressed in small intestine but not in stomach, liver, heart, skeletal muscle, lung, kidney, adrenals, or brain. Bioactivity studies demonstrated that neither gastrotropin nor a carboxyl-terminally amidated tridecapeptide fragment deduced from the cDNA sequence influence acid secretory activity in rats with gastric fistulas or in isolated canine gastric parietal cells. Together these data suggest that gastrotropin is not likely to be secreted as a hormone or to function as an enterooxyntin. Moreover, it appears that gastrotropin represents one of several members of the family of hydrophobic ligand binding proteins that are expressed in the small intestine.
Amino Acid Sequence↗
Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli. Analysis of the apoprotein and the holoprotein containing bound all-trans-retinol and all-trans-retinal.
Rat cellular retinol-binding protein II (CRBP II) is a 15.6-kDa intestinal protein which binds all-trans-retinol and all-trans-retinal but not all-trans-retinoic acid. We have previously analyzed the interaction of Escherichia coli-derived rat apoCRBP II with several retinoids using fluorescence spectroscopic techniques. Interpretation of these experiments is complicated, because the protein has 4 tryptophan residues. To further investigate ligand-protein interactions, we have utilized 19F nuclear magnetic resonance (NMR) spectroscopy of CRBP II labeled at its 4 tryptophan residues with 6-fluorotryptophan. Efficient incorporation of 6-fluorotryptophan (93%) was achieved by growing a tryptophan auxotroph of E. coli harboring a prokaryotic expression vector with a full-length rat CRBP II cDNA on defined medium supplemented with the analog. Comparison of the 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II with and without bound all-trans-retinol revealed that resonances corresponding to 2 tryptophan residues (designated WA and WB) undergo large downfield changes in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand binding. In contrast, 19F resonances corresponding to two other tryptophan residues (WC and WD) undergo only minor perturbations in chemical shifts. The 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II complexed with all-trans-retinal and all-trans-retinol were very similar, suggesting that the interactions of these two ligands with the protein are similar. Molecular model building, based on the crystalline structures of two homologous proteins was used to predict the positions of the 4 tryptophan residues of CRBP II and to make tentative resonance assignments. The fact that ligand binding produced residue-specific changes in the chemical shifts of resonances in CRBP II suggests that NMR analysis of isotopically labeled retinoid-binding proteins expressed in E. coli will provide an alternate, albeit it complementary, approach to fluorescence spectroscopy for examining the structural consequences of their association with ligand.
Amino Acid Sequence↗
Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate.
Rat intestinal fatty-acid-binding protein (I-FABP) is a small (15,124 Mr) cytoplasmic polypeptide that binds long-chain fatty acids in a non-covalent fashion. I-FABP is a member of a family of intracellular binding proteins that are thought to participate in the uptake, transport and/or metabolic targeting of hydrophobic ligands. The crystal structure of Escherichia coli-derived rat I-FABP with a single molecule of bound palmitate has been refined to 2 A resolution using a combination of least-squares methods, energy refinement and molecular dynamics. The combined methods resulted in a model with a crystallographic R-factor of 17.8% (7775 reflections, sigma greater than 2.0), root-mean-square bond length deviation of 0.009 A and root-mean-square bond angle deviation of 2.85 degrees. I-FABP contains ten antiparallel beta-strands organized into two approximately orthogonal, beta-sheets. The hydrocarbon tail of its single C16:0 ligand is present in a well-ordered, distinctively bent conformation. The carboxylate group of the fatty acid is located in the interior of I-FABP and forms a unique "quintet" of electrostatic interactions involving Arg106; Gln 115, and two solvent molecules. The hydrocarbon tail is bent with a slight left-handed helical twist from the carboxylate group to C-16. The bent methylene chain resides in a "cradle" formed by the side-chains of hydrophobic, mainly aromatic, amino acid residues. The refined molecular model of holo-I-FABP suggests several potential locations for entry and exiting of the fatty acid.
Amino Acid Sequence↗
Fatty acid interactions with rat intestinal and liver fatty acid-binding proteins expressed in Escherichia coli. A comparative 13C NMR study.
Enterocytes in the small intestinal mucosa contain abundant quantities of two homologous cytosolic proteins known as intestinal and liver fatty acid-binding proteins (I- and L-FABP, respectively). To elucidate structure-function relationships for these proteins, the interactions between 13C-enriched palmitate and oleate and Escherichia coli-expressed rat I- and L-FABP were systematically compared using 13C NMR spectroscopy. NMR spectra of samples containing fatty acids (FA) and I-FABP at different molar ratios (all at pH 7.2 and 37 degrees C) exhibited a single carboxyl resonance corresponding to FA bound to I-FABP (181.4 ppm, peak I) and an additional carboxyl resonance corresponding to unbound FA in a bilayer phase (179.6 ppm). Peak I reached a maximum intensity corresponding to 1 mol of bound FA/mol of I-FABP under all sample conditions examined. NMR spectra for samples containing FA and L-FABP also exhibited a single carboxyl resonance corresponding to FA bound to L-FABP but at a different chemical shift value (182.2 ppm, peak L). Its maximum intensity varied depending on the physical state of the unbound FA (liquid crystalline or crystalline), the FA used (palmitate or oleate), and the sample pH. In the presence of a liquid crystalline (bilayer) phase, up to 1 (oleate) or 2 (palmitate) mol of FA were bound/mol of L-FABP, but in the presence of a crystalline phase (1:1 acid-soap), up to 3 mol of palmitate were bound/mol of L-FABP (all at pH 7.2). Peak I exhibited little or no ionization shift over a wide pH range (pH 3.0-11.0), and its chemical shift was unaffected by the ionization of Lys and His residues. Hence, the carboxylate group of FA bound to I-FABP was solvent inaccessible and most likely involved in an ion-pair electrostatic interaction with the delta-guanidinium moiety of an Arg residue. In contrast, peak L exhibited an ionization shift and an estimated apparent pKa value similar to that obtained for monomeric FA in water, suggesting that the carboxylate groups of FA bound to L-FABP were solvent accessible and located at or near the protein solvent interface. With decreasing pH, FA dissociated from L-FABP but not I-FABP, as monitored by NMR peak intensities. Concurrently, a large decrease in circular dichroism molar ellipticity was observed with L-FABP but not I-FABP. In conclusion, I-FABP and L-FABP are distinct with regards to their FA-binding stoichiometries, binding mechanisms, and sensitivity to pH.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Refined apoprotein structure of rat intestinal fatty acid binding protein produced in Escherichia coli.
Rat intestinal fatty acid binding protein (I-FABP) is a member of a family of cytoplasmic hydrophobic ligand-binding proteins. To gain insights about the contribution of bound fatty acid to I-FABP's conformation and mechanism of ligand binding, we have determined the structure of Escherichia coli-derived rat apo-I-FABP to 1.96-A resolution and compared it to the recently refined structure of I-FABP with bound palmitate. Both apo- and holo-I-FABP are composed primarily of anti-parallel beta-strands which form two nearly orthogonal beta-sheets ("beta-clam"). The overall structures of the apo- and holo-I-FABP are nearly identical, with a root mean square (rms) difference of 0.37 A between C alpha atoms, 0.38 A between all main-chain atoms, and 0.94 A between all side-chain atoms. However, rms differences of greater than 1.3 A were noted for the side chains of Ile-23, Lys-27, Arg-56, Leu-72, Ala-73, and Asp-74. The space occupied by bound ligand in the core of the holoprotein is occupied in the apo-protein by ordered solvent molecules. This results in an increase in the total number of internal ordered solvent molecules from 7 in the holoprotein to 13 in apo-I-FABP. This finding, together with observed differences in the side-chain orientations of two residues (Arg-56 and Lys-27) situated over a potential opening to the cores of the apo- and holoproteins, suggests that solvent molecules play a critical role in ligand binding. Moreover, the data indicate that the beta-clam structure is stable even in the absence of bound ligand.
Apoproteins↗
Amino acid sequence of porcine heart fumarase.
The complete amino acid sequence of porcine heart fumarase (EC 4.2.1.2) has been determined from peptides produced by cyanogen bromide, endoproteinase Arg-C, S. aureus V8 protease, and trypsin. The enzyme is a tetramer of identical subunits with Mr = 50,015 and composed of 466 amino acid residues. Porcine heart fumarase displays 96% identity to human liver fumarase. Prediction of the secondary structural elements of porcine fumarase indicate that the enzyme contains a large amount of alpha helix with very little beta structure.
Amino Acid Sequence↗
The structure of crystalline Escherichia coli-derived rat intestinal fatty acid-binding protein at 2.5-A resolution.
Rat intestinal fatty acid-binding protein (I-FABP) is an abundant cytoplasmic protein which is synthesized in the small intestinal lining cell where it is thought to participate in the absorption and intracellular metabolism of fatty acids. Each mole of this 132-residue polypeptide binds 1 mol of long chain fatty acid in a noncovalent fashion. Because of its small size and single ligand-binding site, I-FABP represents an attractive model for defining the molecular details of long chain fatty acid-protein interactions. The structure of Escherichia coli-derived rat I-FABP has now been solved to 2.5 A resolution using three isomorphous heavy atom derivatives. The protein consists of 10 anti-parallel beta-strands present as two orthogonal beta-sheets. Together a "clam shell-like" structure is formed with an opening located between two beta-strands and an interior that is lined with the side chains of nonpolar amino acids. The bound fatty acid ligand is located in the interior of the protein and has a bent conformation, possibly reflecting the presence of several gauche bonds in the hydrocarbon tail. Our present interpretation of the electron density map suggests that the fatty acid is oriented with its carboxylate group facing the guanidinium group of Arg127, whereas the end of its hydrocarbon tail is in close proximity to Val106. The indole side chain of Trp83 forms the molecular framework around which the principal bend of the hydrocarbon chain occurs.
Amino Acid Sequence↗
Crystallization of rat cellular retinol binding protein II. Preliminary X-ray data obtained from the apoprotein expressed in Escherichia coli.
Rat cellular retinol-binding protein II (CRBP II) is a member of a family of cytoplasmic proteins which bind hydrophobic ligands. CRBP II is thought to participate in the intestinal absorption and intracellular metabolism of retinoids. We have previously described the crystallization of a homologous rat intestinal fatty acid-binding protein (I-FABP) isolated from Escherichia coli containing a suitably constructed prokaryotic expression vector (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J., J. Biol. Chem. 262, 5428-5430). We have now efficiently expressed rat CRBP II in E. coli. The E. coli-derived protein, which does not contain any bound retinoid, has been purified and crystals grown from solutions of polyethylene glycol 4000. Crystals of apo-CRBP II are triclinic, space group P1, a = 36.8 A, b = 64.0 A, c = 30.4 A; alpha = 92.8 degrees, beta = 113.5 degrees, gamma = 90.1 degrees. Each unit cell contains two molecules of the 134-residue apoprotein. X-ray diffraction data suggest that the unit cell parameters of crystalline apo-CRBP II resemble those of I-FABP. Comparison of the tertiary structures of E. coli-derived rat I-FABP and CRBP II should provide insights about how these proteins evolved to bind different hydrophobic ligands.
Animals↗
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.
Amino Acid Sequence↗
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.
Animals↗