Thermodynamics of fatty acid binding to engineered mutants of the adipocyte and intestinal fatty acid-binding proteins

• Published in: The Journal of biological chemistry 1998-03, Vol.273 (13), p.7397-7405
• Main Authors: Richieri, G.V, Low, P.J, Ogata, R.T, Kleinfeld, A.M
• Format: Article
• Language: English
• Subjects: ACIDE GRAS; ACIDOS GRASOS; Adipocytes - metabolism; ADIPOSE TISSUES; Amino Acid Substitution; Animals; BINDING PROTEINS; BINDING SITES; Carrier Proteins - genetics; Carrier Proteins - metabolism; CELLS; CELLULE; CELULAS; DISSOCIATION; ENERGIA; ENERGIE; ENERGY; ENTHALPY; ENTROPY; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; FATTY ACIDS; Fatty Acids - metabolism; FISICA; FREE ENERGY; GENERO HUMANO; GENRE HUMAIN; INDUCED MUTATION; INTESTIN; Intestinal Mucosa - metabolism; INTESTINES; INTESTINOS; Kinetics; MANKIND; Models, Molecular; MUTACION; MUTACION INDUCIDA; MUTANT; MUTANTES; MUTANTS; MUTATION; MUTATION PROVOQUEE; Myelin P2 Protein - genetics; Myelin P2 Protein - metabolism; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid - metabolism; PHYSICS; PHYSIQUE; Protein Engineering; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; Rats; Structure-Activity Relationship; TARGETED MUTAGENESIS; TEJIDO ADIPOSO; Thermodynamics; TISSU ADIPEUX
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.13.7397

We constructed 18 single amino acid mutants of the adipocyte fatty acid-binding protein (A-FABP) and 17 of the intestinal fatty acid-binding protein (I-FABP), at locations in the fatty acid (FA) binding sites. For each mutant protein, we measured thermodynamic parameters that characterize FA binding. Binding affinities ranged from about 200-fold smaller to 30-fold larger than the wild type (WT) proteins. Thermodynamic parameters revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory and larger than the binding free energy. FA-FABP interactions were quite different for I-FABP and A-FABP proteins. Binding affinities were larger and decreased to a greater degree with increasing FA solubility for most of the I-FABP as compared with the A-FABP proteins, consistent with a more hydrophobic binding site for the I-FABP proteins. In A-FABP, Ala substitutions for Arg 106 and Arg 126 , which interact with the FA carboxylate, reduce affinities by about 100-fold, but in I-FABP, R106A increases affinities up to 30-fold. Moreover, in A-FABP, the thermodynamic parameters predict that the FA carboxylate location switches from the 126-position in R106A to the 106 position in R126A. Finally, the A-FABP proteins, in contrast to the I-FABP proteins, reveal significant heat capacity changes (Δ C p ) upon FA binding, and substitutions at residues Arg 106 and Arg 126 reduce the magnitude of Δ C p .