Biophysical Approaches for Investigating the Dynamics and Cooperativity of Ternary Complexes in Targeted Protein...

Heterobifunctional small molecules (hSMs) are a rapidly emerging therapeutic modality that induces spatial proximity between biomolecules, such as proteins, by forming a ternary complex. In this study, we introduce biophysical methodologies to study ternary complexes involving hydrodynamic profiling by nuclear magnetic resonance spectroscopy (HAP-NMR) and isothermal titration calorimetry (ITC). These approaches allowed us to evaluate the hydrodynamic and thermodynamic factors that influence molecular dynamics and ternary complex formation in solution. We addressed challenges like the limited solubility of hSMs, particularly in ITC experiments, and the near-ideal equimolar stoichiometry (1:1:1) of the ternary complex in solution, which is necessary for accurate hydrodynamic parameter evaluation. Our methodology facilitated the characterization of a known degrader of SMARCA proteins and revealed subtle higher-level structural differences. This approach provided insights into the overall behavior and function of the SMARCA2 and SMARCA4 protein ternary complexes.