Functional thermodynamics govern the ligand binding to human cytosolic transport proteins.

Traditional binding studies on cellular, molecular transporters often simplify the thermodynamics of binding processes. We present a method that combines spin probing, electron paramagnetic resonance (EPR) spectroscopy and spectral simulations with binding studies and thermodynamics for an in-depth molecular view into ligand binding and protein functionality. We prove this approach by studying the temperature-dependent thermodynamics of fatty acids binding to a family of human cytoplasmic transport and signaling proteins, fatty acid binding proteins FABP3, FABP4 and FABP5. The proteins were loaded with the radical-bearing ligands 5- and 16-DOXYL stearic acid (5/16-DSA) and continuous-wave (CW) EPR spectroscopy was applied in a broad temperature range. In this methodology, originally developed by us to study ligand binding to synthetic functional polymers, spectral simulations provide the concentrations and equilibria of free, intermediately, and strongly bound ligands. The derivation of temperature-dependent binding affinities and thermodynamic parameters enables the simultaneous analysis of multiple binding processes. We found and elucidate a thermodynamic preference for loose, entropy-driven attachment at physiological conditions. The approach delivered complex, temperature-dependent binding thermodynamics, revealing similarities and discrepancies between various FABP isoforms and concentration regimes. This corroborates the hypothesis that these transport proteins evolved functional thermodynamics with fine-tuned thermodynamic binding profiles to fulfill respective physiological functionalities. The study delivers important information about protein-ligand interactions in general and establishes EPR-based thermodynamic analyses as a platform to study and tune native or synthetic polymeric transport systems for advanced applications.