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

ABSTRACT: 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.

SUBMITTER: He Y

PROVIDER: S-EPMC3072248 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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

He Yan Y Estephan Rima R Yang Xiaomin X Vela Adriana A Wang Hsin H Bernard Cédric C Stark Ruth E RE

Biochemistry 20110202 8

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 det ...[more]

PMID: 21226535

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Project description:Background Supplementation with omega-3 (n-3) fatty acid or dietary fish may protect against atherosclerosis, but the potential mechanisms are unclear. Prior studies found modest triglyceride-lowering effects and slight increases in LDL (low-density lipoprotein) cholesterol. Limited evidence has examined n-3 effects on more detailed lipoprotein biomarkers. Methods and Results We conducted a study of 26 034 healthy women who reported information on fish and n-3 intake from a 131-item food-frequency questionnaire. We measured plasma lipids, apolipoproteins, and nuclear magnetic resonance spectroscopy lipoproteins and examined their associations with dietary intake of fish, total n-3, and the n-3 subtypes (eicosapentaenoic, docosahexaenoic, and α-linolenic acids). Top- versus bottom-quintile intake of fish and n-3 were significantly associated with lower triglyceride and large VLDL (very-low-density lipoprotein) particles. Fish intake, but not total n-3, was positively associated with total cholesterol, LDL cholesterol, apolipoprotein B, and larger LDL size, but only α-linolenic acid was associated with lower LDL cholesterol. Total n-3, docosahexaenoic acid, and α-linolenic acid intake were also positively associated with larger HDL (high-density lipoprotein) size and large HDL particles. High eicosapentaenoic acid intake was significantly associated with only a decreased level of VLDL particle concentration and VLDL triglyceride content. The n-3 fatty acids had some similarities but also differed in their associations with prospective cardiovascular disease risk patterns. Conclusions Higher consumption of fish and n-3 fatty acids were associated with multiple measures of lipoproteins that were mostly consistent with cardiovascular prevention, with differences noted for high intake of eicosapentaenoic acid versus docosahexaenoic acid and α-linolenic acid that were apparent with more detailed lipoprotein phenotyping. These hypothesis-generating findings warrant further study in clinical trials. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT00000479.

Project description:In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Dataset Information

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

Publications

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

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