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 Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein.

1991 ◽  
Vol 266 (9) ◽  
pp. 5486-5496
Author(s):  
J R Jefferson ◽  
J P Slotte ◽  
G Nemecz ◽  
A Pastuszyn ◽  
T J Scallen ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
L Cell

scholarly journals Effect on ligand binding of arginine mutations in recombinant rat liver fatty acid-binding protein

1994 ◽  
Vol 297 (1) ◽  
pp. 103-107 ◽  
Author(s):  
A E Thumser ◽  
C Evans ◽  
A F Worrall ◽  
D C Wilton
Keyword(s):  
Fatty Acid ◽  
Ligand Binding ◽  
Rat Liver ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Wide Range ◽  
Arginine Residues

Rat liver fatty acid-binding protein is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. The two arginine residues of rat liver fatty acid-binding protein, Arg122 and Arg126, have been mutated and the effect of mutation on ligand binding investigated. No significant decrease in affinity for the fluorescent fatty acid analogue, 11-(5-dimethylaminonaphthalenesulphonyl amino)undecanoic acid, or oleate was observed. However, the apparent affinity for oleoyl-CoA was slightly increased with the mutations Ala122 and Gln122 such that oleoyl-CoA rather than oleate became the preferred ligand for these mutants. Small changes in protein stability were observed with the Arg122 mutations. The lack of notable ionic involvement of the conserved internal residue Arg122 in ligand binding is consistent with the hypothesis that the mode of ligand binding in liver fatty acid-binding protein is markedly different from that of other members of this lipid-binding protein family.

scholarly journals The binding of natural and fluorescent lysophospholipids to wild-type and mutant rat liver fatty acid-binding protein and albumin

1995 ◽  
Vol 307 (1) ◽  
pp. 305-311 ◽  
Author(s):  
A E A Thumser ◽  
D C Wilton
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Kd Values ◽  
Fluorescence Characteristics ◽  
Wide Range

Rat liver fatty acid-binding protein (FABP) is able to bind a wide range of non-polar anionic ligands, including lysophospholipids. In order to understand the nature of lysophospholipid interactions with liver FABP, the binding of natural lysophospholipids and two fluorescent analogues, N-(5-dimethylaminonaphthalenesulphonyl)-1-palmitoyl-sn-glycero-3- phosphoethanolamine (dansyl lysoPE) and 1-(O-[11-(5-dimethylaminonaphthalene-sulphonyl)amino]undecyl)-sn-glycero -3- phosphocholine (dansyl-C11-lysoPAF), has been investigated. The results confirmed the ability of liver FABP to bind lysophospholipids with KD values in the range of 1-2 microM, and a 1:1 binding stoichiometry was indicated. Binding of fluorescent lysophospholipids was enhanced with the FABP mutant, R122Q, possibly due to increased flexibility of the binding cavity as a result of reduced hydrogen-bonding constraints. The fluorescent lysophospholipids also bound to albumin, with KD values in the range 0.1-1.0 microM, and could be displaced by oleic acid. The fluorescence characteristics of the dansyl lysophospholipid analogue dansyl-C11-lyso-PAF suggested that this probe binds to the same site(s) on albumin as the fluorescent fatty acid probe 11-(5-dimethylaminonaphthalene-sulphonylamino)-undecanoic acid (DAUDA).

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