The three‐dimensional structure of the holo form of recombinant cellular bovine heart fatty‐acid‐binding protein (H‐FABPc), a polypeptide of 133 amino acid residues with a molecular mass of 15 kDa, has been determined by multidimensional homonuclear and heteronuclear NMR spectroscopy applied to uniformly 15N‐labeled and unlabeled protein. A nearly complete set of 1H and 15N chemical shift assignments was obtained. A total of 2329 intramolecular distance constraints and 42 side‐chain χi dihedral‐angle constraints were derived from cross‐relaxation and J coupling information. 3D nuclear Overhauser enhancement and exchange spectroscopy combined with heteronuclear multiple‐quantum coherence (NOESY‐HMQC) experiments, performed on a sample of uniformly 13C‐labeled palmitic acid bound to unlabeled cellular heart fatty‐acid‐binding protein revealed 10 intermolecular contacts that determine the orientation of the bound fatty acid. An ensemble of protein conformations was calculated with the distance‐geometry algorithm for NMR applications (DIANA) using the redundant dihedral‐angle constraint (REDAC) strategy. After docking the fatty acid into the protein, the protein‐ligand arrangement was subject to distance‐restrained energy minimization. The overall conformation of the protein is a β‐barrel consisting of 10 antiparallel β‐strands which form two nearly orthogonal β‐sheets of five strands each. Two short helices form a helix‐turn‐helix motif in the N‐terminal region of the polypeptide chain. The palmitic acid is bound within the protein in a U‐shaped conformation close to the two helices. The obtained solution structure of the protein is consistent with a number of fatty‐acid‐binding‐protein crystal structures.

Two-and three-dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty-acid-binding protein (heart FABP). NOE and rotating-frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two-dimensional H 2O-ROE/NOE-1 H, 15 N-heteronuclear single quantum coherence spectra recorded from a uniformly 13 C/ 15 N-enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel β-strands (βAϪβJ), forming two nearly orthogonal β-sheets, and a short helixturn-helix motif connecting β-strands A and B. The spatial folding resembles a β-barrel. Most of the water molecules are localized in the gap between β-strands D and E, and near the two A-helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three-dimensional rotating frame Overhauser 1 H, 15 N heteronuclear single quantum coherence experiment obtained with a 15 N-labeled sample of apo-heart FABP.Keywords : bound water; apoprotein ; holoprotein; fatty-acid-binding protein; NMR.Fatty-acid-binding proteins (FABPs) belong to a class of cy-large cavity. Although the conformations of bound ligands differ tosolic, non-enzymic proteins with high non-covalent affinity for in the distinct FABP types, the conformation of the apo form of long-chain fatty acids and molecular masses of 14Ϫ15 kDa [1, the protein is very similar to the holo form [14]. 2]. FABPs have been isolated from a number of fatty-acid-me-A solution structure of bovine heart FABP has been pretabolizing tissues of mammals, fish, chicken, and insect [2]. It sented previously [15Ϫ17]. The fatty acid ligand is bound in a is proposed that fatty-acid-binding proteins serve as a pool for U-shaped conformation, within the binding cavity [17]. The solubilized fatty acids and protect the cells from the detergent crystal structure of the homologous heart FABP of human museffects of high fatty acid concentrations [3]. They are involved cle with bound fatty acid revealed 13 water molecules within in the cellular uptake of fatty acids and in the intracellular trans-this cavity. Two of these water molecules interact with the carport of fatty acids to the mitochondria. They may modulate en-boxylate group of the fatty acid. A mechanism for fatty acid zyme activities and have an influence on cell growth and cell binding is proposed by Young and coworkers [5] including the differentiation [2]. Nevertheless the precise physiological func-rearrangement of some sidechains and water molecules. Calculation of FABP still remains to be elucidated.tions of the solvent-accessible surface of human muscle heart At present seven distinct FABP types are known which have FABP suggest that the internal cavity is connected to the exterbeen isolated from liver, intestine, heart, adipocyte, myelin-type nal solvent by a portal area w...

The nearly complete sequence‐specific 1H resonance assignment of the pI = 4.9 isoform of cytosolic 15‐kDa fatty‐acid‐binding protein from bovine heart (H‐FABPc) by homonuclear two‐dimensional NMR spectroscopy is presented. Regular secondary structure elements were identified from NOE spectra and the sequence locations of slowly exchanging backbone amide protons. The molecular structure of the protein was found to consist mainly of ten antiparallel β‐strands and two short α‐helices. The data presented here for the first time for a hydrophobic molecule transporter of the fatty‐acid‐binding protein type is the basis for a complete tertiary structure determination currently in progress.

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The three-dimensional structure of the holo form of recombinant cellular bovine heart fatty-acid-binding protein (H-FABPc), a polypeptide of 133 amino acid residues with a molecular mass of 15 kDa, has been determined by multidimensional homonuclear and heteronuclear NMR spectroscopy applied to uniformly 15N-labeled and unlabeled protein. A nearly complete set of 1H and 15N chemical shift assignments was obtained. A total of 2329 intramolecular distance constraints and 42 side-chain chi 1 dihedral-angle constraints were derived from cross-relaxation and J coupling information. 3D nuclear Overhauser enhancement and exchange spectroscopy combined with heteronuclear multiple-quantum coherence (NOESY-HMQC) experiments, performed on a sample of uniformly 13C-labeled palmitic acid bound to unlabeled cellular heart fatty-acid-binding protein revealed 10 intermolecular contacts that determine the orientation of the bound fatty acid. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DIANA) using the redundant dihedral-angle constraint (REDAC) strategy. After docking the fatty acid into the protein, the protein-ligand arrangement was subject to distance-restrained energy minimization. The overall conformation of the protein is a beta-barrel consisting of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each. Two short helices form a helix-turn-helix motif in the N-terminal region of the polypeptide chain. The palmitic acid is bound within the protein in a U-shaped conformation close to the two helices. The obtained solution structure of the protein is consistent with a number of fatty-acid-binding-protein crystal structures.