Three-dimensional structure of recombinant human muscle fatty acid-binding protein.

Human muscle fatty acid-binding protein (M-FABP) is a 15 kDa cytosolic protein which may be involved in fatty acid transfer and modulation of non-esterified fatty acid concentration in heart, skeletal muscle, kidney and many other tissues. Crystallographic studies have suggested the importance of the amino acids Thr-40, Arg-106, Arg-126 and Tyr-128 for the hydrogen bonding network of the fatty acid carboxylate group. Two phenylalanines at 16 and 57 are positioned to interact with the acyl chain of the fatty acid. We prepared 13 mutant proteins by site-directed mutagenesis and tested them for fatty acid binding and stability. Substitution of amino acids Phe-16, Arg-106 or Arg-126 created proteins which showed a large decrease in or complete loss of oleic acid binding. Substitution of Phe-57 by Ser or Val and of Tyr-128 by Phe had no great effect. The stability of the mutant proteins was tested by denaturation studies on the basis of fatty acid binding or tryptophan fluorescence and compared with that of the wild-type M-FABP. There was no direct relationship between fatty acid-binding activity and stability. Less stable mutants (F57S and Y128F) did not show a marked change in fatty acid-binding activity. Substitution of Arg-126 by Gln or Arg-106 by Thr eliminated binding activity, but the former mutant protein showed wild-type stability, in contrast to the latter. The results are in agreement with crystallographic data.

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Three-Dimensional Structure of Bovine Heart Fatty-acid-binding Protein with Bound Palmitic Acid, Determined by Multidimensional NMR Spectroscopy

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.tb20560.x ◽

1995 ◽

Vol 230 (1) ◽

pp. 266-280 ◽

Cited By ~ 28

Author(s):

Dirck Lassen ◽

Christian Lucke ◽

Marina Kveder ◽

Azita Mesgarzadeh ◽

Jurgen M. Schmidt ◽

...

Keyword(s):

Fatty Acid ◽

Nmr Spectroscopy ◽

Palmitic Acid ◽

Fatty Acid Binding Protein ◽

Three Dimensional ◽

Dimensional Structure ◽

Fatty Acid Binding ◽

Acid Binding Protein ◽

Bovine Heart ◽

Multidimensional Nmr Spectroscopy

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Identification of a non-classical three-dimensional nuclear localization signal in the intestinal fatty acid binding protein

PLoS ONE ◽

10.1371/journal.pone.0242312 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0242312

Author(s):

Mariana Suárez ◽

Lucía Canclini ◽

Adriana Esteves

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Binding Protein ◽

Nuclear Translocation ◽

Binding Protein ◽

Three Dimensional ◽

Long Chain Fatty Acids ◽

Fatty Acid Binding ◽

Acid Binding Protein ◽

Localization Signal

The intestinal fatty acid binding protein (FABP) is a small protein expressed along the small intestine that bind long-chain fatty acids and other hydrophobic ligands. Several lines of evidence suggest that, once in the nucleus, it interacts with nuclear receptors, activating them and thus transferring the bound ligand into the nucleus. Previous work by our group suggests that FABP2 would participate in the cytoplasm-nucleus translocation of fatty acids. Because the consensus NLS is absent in the sequence of FABP2, we propose that a 3D signal could be responsible for its nuclear translocation. The results obtained by transfection assays of recombinant wild type and mutated forms of Danio rerio Fabp2 in Caco-2 cell cultures, showed that lysine 17, arginine 29 and lysine 30 residues, which are located in the helix-turn-helix region, would constitute a functional non-classical three-dimensional NLS.

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Expression in Escherichia coli and characterization of the fatty-acid-binding protein from human muscle

Biochemical Journal ◽

10.1042/bj2780361 ◽

1991 ◽

Vol 278 (2) ◽

pp. 361-364 ◽

Cited By ~ 17

Author(s):

R A Peeters ◽

J M Ena ◽

J H Veerkamp

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Fatty Acid Binding Protein ◽

Unsaturated Fatty Acids ◽

Binding Protein ◽

Exchange Chromatography ◽

Human Muscle ◽

Complete Agreement ◽

Fatty Acid Binding ◽

Acid Binding Protein

The coding part of the cDNA encoding human muscle fatty-acid-binding protein (FABP) was ligated in the pET8c vector and expressed under the control of the lacUV5 promoter. After induction with isopropyl beta-D-thiogalactopyranoside, almost 12% of the cytoplasmic proteins consisted of FABP. The protein could be isolated after sonication of the bacterial pellet followed by (NH4)2SO4 precipitations, anion-exchange chromatography and gel filtration. The muscle FABP produced in Escherichia coli has an isoelectric point of 5.3 and is recognized by anti-(human muscle FABP) antiserum after Western blotting. The purified FABP has a preference for binding to palmitic acid and C18-C22 (poly)unsaturated fatty acids, and no affinity to palmitoyl-CoA or other hydrophobic ligands tested. The dissociation constant for oleic acid is 0.58 microM, with a binding stoichiometry of 0.72 mol of fatty acid/mol of protein. The physicochemical and binding characteristics of the protein were in complete agreement with those of FABP isolated from human skeletal muscle.

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