Thermodynamics of fatty acid binding to engineered mutants of the adipocyte and intestinal fatty acid-binding proteins.
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 Arg106 and Arg126, 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 (DeltaCp) upon FA binding, and substitutions at residues Arg106 and Arg126 reduce the magnitude of DeltaCp.
Richieri
G V
GV
Medical Biology Institute, La Jolla, California 92037, USA.
Low
P J
PJ
Ogata
R T
RT
Kleinfeld
A M
AM
D017667:Adipocytes / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018996:Myelin P2 Protein / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid / Q000378:metabolism; D015202:Protein Engineering; D051381:Rats; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid