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Fatty acid interactions with native and mutant fatty acid binding proteins
G V Richieri1, R T Ogata, A M Kleinfeld
1Medical Biology Institute, La Jolla, CA, USA.
Molecular and Cellular Biochemistry
|May 20, 1999
Summary
Fatty acid binding proteins (FABPs) buffer intracellular free fatty acids (FFAs), maintaining concentration gradients for transport. Binding affinities vary by FABP type and fatty acid solubility, with rapid equilibrium achieved within seconds.
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.