Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty a cid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constan ts for binding and dissociation. These measurements have been done using th e fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of t he free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins f rom adipocyte, heart, intestine, and liver, K-d values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Bindi ng affinities for heart and liver are generally greater than those for adip ocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37 degrees C is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FF A, suggests a buffering action of FABPs that helps to maintain the intracel lular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature depen dence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of th e intestinal FABP. These measurements showed that binding affinities did no t report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an ala nine substitution for arginine 106 yields a 30 fold increase in binding aff inity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg(106), is more than compensa ted for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.