Fatty acid interactions with native and mutant fatty acid binding proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Physiology

Background:

• Fatty acid binding proteins (FABPs) are crucial for intracellular fatty acid (FA) transport and metabolism.
• Understanding FA-FABP interactions is key to comprehending cellular lipid homeostasis and signaling.

Purpose of the Study:

• To quantify binding equilibrium and kinetics of long-chain fatty acids (FA) with wild-type (WT) and mutant fatty acid binding proteins (FABPs).
• To elucidate the thermodynamic basis of FA-FABP interactions and the role of specific amino acid residues.

Main Methods:

• Utilized fluorescent probes ADIFAB and ADIFAB2 to measure free fatty acid (FFA) concentrations.
• Determined equilibrium binding constants (Kd) and rate constants for binding and dissociation.
• Investigated temperature dependence of binding and thermodynamics of interactions with engineered FABP mutants.

Main Results:

• WT FABPs (adipocyte, heart, intestine, liver) exhibit nM binding affinities (Kd) for FAs, decreasing with increased FA aqueous solubility.
• Heart and liver FABP affinities generally exceed those of adipocyte and intestine FABPs.
• Binding equilibrium is rapidly achieved (within seconds) at 37°C.
• Thermodynamic analysis reveals predominantly enthalpic contributions to binding free energy, driven by FA-FABP interactions.
• Mutational analysis shows compensatory changes in enthalpy and entropy, where affinity changes do not always reflect direct interaction modifications.

Conclusions:

• FABPs act as intracellular buffers for FFAs, maintaining concentration gradients essential for cellular uptake and release.
• FA-FABP interactions are primarily enthalpic, stemming from specific contacts within the binding cavity.
• Accurate characterization of FA-FABP interactions necessitates evaluating both enthalpy and entropy, not just binding affinity, due to compensatory effects in mutants.