scholarly journals Fatty acid binding and conformational stability of mutants of human muscle fatty acid-binding protein

1996 ◽  
Vol 314 (1) ◽  
pp. 253-260 ◽  
Author(s):  
Clemens F. M. PRINSEN ◽  
Jacques H. VEERKAMP
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Binding Activity ◽  
Human Muscle ◽  
Wild Type ◽  
Fatty Acid Binding ◽  
Mutant Proteins ◽  
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.

Structural studies on human muscle fatty acid binding protein at 1.4 å resolution: binding interactions with three C18 fatty acids

Structure ◽  
1994 ◽  
Vol 2 (6) ◽  
pp. 523-534 ◽  
Author(s):  
Aideen CM Young ◽  
Giovanna Scapin ◽  
Arno Kromminga ◽  
Sangita B Patel ◽  
Jacques H Veerkamp ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Human Muscle ◽  
Structural Studies ◽  
Binding Interactions ◽  
Fatty Acid Binding ◽  
Acid Binding Protein

scholarly journals Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants

1994 ◽  
Vol 300 (3) ◽  
pp. 827-833 ◽  
Author(s):  
A E Thumser ◽  
D C Wilton
Keyword(s):  
Energy Transfer ◽  
Fatty Acid ◽  
Fluorescence Quenching ◽  
Rat Liver ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Mutant Proteins ◽  
Acid Binding Protein

Rat liver fatty-acid-binding protein (FABP) does not contain tryptophan. Three mutant proteins have been produced in which a single tryptophan residue has been inserted by site-directed mutagenesis at positions 3 (F3W), 18 (F18W) and 69 (C69W). These tryptophans have been strategically located in order to provide fluorescent reporter groups to study the binding and structural characteristics of rat liver FABP. Two fluorescent fatty acid analogues, DAUDA (11-[(5-dimethylaminonaphthalene-1- sulphonyl)amino]undecanoic acid) and 3-[p-(6-phenyl)-hexa-1,3,5-trienyl]phenylpropionic acid, showed no significant difference in binding affinities for the different mutant proteins, although maximum fluorescence values were decreased for F3W and increased for C69W. These findings were confirmed by studies of DAUDA displacement by oleate. Protein-denaturation studies in the presence of urea indicated subtle differences for the three mutants which could be explained by multiple unfolding pathways. Fatty acid binding increased tryptophan fluorescence emission in the case of the F18W protein, but had no effect on the F3W and C69W proteins. Fluorescence quenching studies with 2-bromopalmitate showed that a fatty acid carboxylate is close to the tryptophan in the F18W protein. Energy-transfer studies showed that the fluorescent moiety of DAUDA is equidistant from the three mutated amino acids and is bound within the beta-clam solvent cavity of liver FABP. This interpretation of the fluorescence quenching and energy-transfer data supports the difference in ligand orientation between intestinal and liver FABP observed in previous studies.

scholarly journals Codon-54 Polymorphism of the Fatty Acid-Binding Protein 2 Gene Is Associated with Elevation of Fasting and Postprandial Triglyceride in Type 2 Diabetes*

2000 ◽  
Vol 85 (9) ◽  
pp. 3155-3160 ◽  
Author(s):  
Angeliki Georgopoulos ◽  
Omer Aras ◽  
Michael Y. Tsai
Keyword(s):  
Type 2 Diabetes ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Plasma Triglyceride ◽  
Wild Type ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Fasting Plasma

Abstract Patients with type 2 diabetes are frequently dyslipidemic or hypertriglyceridemic. To assess whether increased intestinal triglyceride input leads to elevated fasting and postprandial triglycerides in type 2 diabetes, we used the codon 54 polymorphism of the fatty acid-binding protein 2 gene, which results in the substitution of threonine (Thr) for alanine and is associated with increased intestinal input of triglyceride. Of the 287 diabetic patients screened, 108 (37.6%) were heterozygous and 31 (10.8%) were homozygous for the Thr-54 allele. Mean (±sem) fasting plasma triglyceride levels in patients with the wild-type (n = 80), those heterozygous for the Thr-54 allele (n = 57), and those homozygous for it (n = 18) were 2.0 ± 0.09, 2.7 ± 0.20, and 3.8 ± 0.43 mmol/L, respectively. A linear relationship of mean fasting plasma triglyceride levels (r2 = 0.97) between the 3 groups was found. After fat ingestion, the postprandial area under the curve of plasma triglyceride (P = 0.025) and chylomicrons (Sf > 400, P = 0.013) was higher in the Thr-54/Thr-54 (n = 6) than in the wild-type (n= 9). Our results are consistent with the hypothesis that, in type 2 diabetes, increased intestinal input of triglyceride can lead to elevated fasting and postprandial plasma triglycerides.

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

1991 ◽  
Vol 278 (2) ◽  
pp. 361-364 ◽  
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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