Abstract
Site-specific variants of rat intestinal fatty acid-binding protein were constructed to identify the molecular interactions that are important for binding to fatty acids (FAs). Several variants displayed affinities that appeared incompatible with the crystal structure of the protein-FA complex. Thermodynamic measurements provided an explanation for these apparent inconsistencies and revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory. These results demonstrate that understanding the effects of amino acid replacements on ligand binding requires measurements of enthalpy and entropy, in addition to affinity.
Highlights
Fatty acid-binding proteins (FABPs)1 are approximately 15kDa cytosolic proteins that may play important roles in fatty acid (FA) trafficking [1,2,3]
To understand how the amino acid residue-FA interactions revealed by the crystal structure contribute to the energy of binding we have used site-specific mutagenesis to alter amino acid residues within the binding cavity
We substituted Ala for 16 of the 19 residues interacting with the FA in the cavity (Fig. 1A), as well as Gln at residue 106
Summary
Fatty acid-binding proteins (FABPs)1 are approximately 15kDa cytosolic proteins that may play important roles in fatty acid (FA) trafficking [1,2,3]. From the temperature dependence of the Kd values we determined the differences in enthalpy (DDH0), entropy (TDDS0), and free energy (DDG0) of FA binding between each mutant and the WT protein.
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