BPS2026 – Functional thermodynamics govern the ligand interactions with human cytosolic transport proteins

How would nature engineer a biophysical transporter that needs to operate with the highest thermodynamic efficiency? To understand the functionalities of water-soluble transport proteins in human cells we propose that it is a viable strategy to analyze their primary function from a thermodynamic perspective. 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 interacting with a family of human cytosolic transport 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. Spectral simulations provide the concentrations and equilibria of freely, intermediately, and strongly bound ligands. The derivation of temperature-dependent binding affinities and thermodynamic parameters enables the simultaneous analysis of multiple interaction processes. The probing of the FABP-ligand interactions is complemented by EPR-based sensing of binding pocket polarities and supported by microscale thermophoresis (MST) and docking simulations. We found and elucidate a thermodynamic preference for entropy-driven, intermediate pre-binding at physiological conditions. The approach delivered complex, temperature-dependent binding thermodynamics, revealing similarities and discrepancies between FABP isoforms from different tissues. This corroborates the hypothesis that these transport proteins evolved adaptive functional thermodynamics with fine-tuned thermodynamic binding profiles to fulfill respective physiological functionalities. The study unifies the thermodynamic and molecular description of 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.