Integrative biophysical characterization of molecular interactions: A case study with the sfGFP–nanobody complex

This study compares several analytical biophysical methods for investigating protein-protein interactions (PPIs) in solution, using the interaction between superfolder green fluorescent protein (sfGFP) and its anti-sfGFP nanobody enhancer as a model system. Techniques evaluated include microscale thermophoresis, fluorescence correlation spectroscopy, analytical ultracentrifugation with multi-wavelength and fluorescence detection, isothermal titration calorimetry, and analytical size exclusion chromatography coupled to multi-angle static light scattering and dynamic light scattering. Each method was assessed for information content, dynamic range, precision, and complementarity. The results consistently indicate a single-digit nanomolar dissociation constant and 1:1 stoichiometry for the interaction. While each technique offers unique insights into binding affinity, thermodynamics, and stoichiometry of the interaction, the multi-method approach provides a more complete and reliable characterization of PPIs. The study demonstrates how combining multiple complementary techniques enhances the robustness of PPI analysis in solution-phase conditions.