Surface Plasmon Resonance-Based Characterization of PROTAC-Induced Ternary Complexes Involving CDK2 and CRBN-DDB1

PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that promote targeted protein degradation by recruiting an E3 ubiquitin ligase to the protein of interest (POI), forming a ternary complex that facilitates ubiquitination and proteasomal degradation. The dynamic nature of these complexes presents challenges for characterization. Conventional methods such as fluorescence polarization (FP), time-resolved FRET (TR-FRET), and AlphaLISA lack real-time and label-free kinetic resolution, while isothermal titration calorimetry (ITC) demands large sample quantities and low throughput. Here, we utilize surface plasmon resonance (SPR) to characterize the binding affinity and cooperativity of a ternary complex comprising cyclin-dependent kinase 2 (CDK2), the PROTAC CPS2, and the CRBN–DDB1 E3 ligase complex. Using the Biacore T200 system, we measure real-time, label-free binary and ternary binding kinetics, revealing a cooperativity factor (α) of approximately 98, indicative of strong positive cooperativity. Our findings highlight SPR as a robust platform for quantitative assessment of PROTAC-induced ternary complexes.