Kinetic and thermodynamic analysis of PKA-R-cAMP interactions in crude media using focal molography

Quantifying biomolecular interactions under near-physiological conditions is essential for understanding biological processes. However, this remains a challenge in established techniques: for surface plasmon resonance, bulk refractive-index shifts and non-specific adsorption in hydrogel matrices can dominate the signal in serum, whereas for isothermal titration calorimetry, complex media primarily introduce large, composition-dependent background heats and run-to-run variability that obscure the binding enthalpy. Here, we demonstrate that focal molography, a label-free optical biosensing method, can reliably measure the kinetics and thermodynamics of the protein kinase A regulatory subunit binding to cyclic AMP derivatives in both buffer and 50% human serum. By performing the kinetic measurement over a range of temperatures, we were able to reveal that binding remains highly favorable in both environments, yet the results suggest that the balance of driving forces shifts toward enthalpic contributions in serum. This hints at the substantial difference of interaction mechanisms in a complex biological media compared to buffer systems. Our findings show that focal molography reliably quantifies protein-ligand interactions in complex media, providing consistent kinetic and thermodynamic data while overcoming limitations of existing methods. This highlights the potential of focal molography as a valuable tool for studying interactions under near-native conditions.