Pulsed Dipolar Spectroscopy (PDS), a branch of Electron Paramagnetic Resonance (EPR) spectroscopy that focuses on the measurement of interspin dipolar interactions, holds significant value for the investigation of protein-protein interactions (PPIs), capable of elucidating structural constraints, determining dynamic information, and counting coupled spins. Protein oligomerization is a specific type of PPI of great interest to pharmaceutical and drug-discovery industries. Because oligomerization influences and regulates many key protein categories such as enzymes, ion channels, and transcription factors, it plays a vital role in many biological and physiological processes that make appealing targets for therapeutic development. Therefore, understanding the mechanism of interaction is of significant interest to the scientific community.
Here, we apply Double Electron Electron Resonance (DEER), a robust and popular PDS technique, to investigate the dimerization of a homodimeric protein system. The distance distributions extracted from the DEER data are used to gain insight into the structure and dynamics of the dimeric complex, while careful analysis of the modulation depth of the dipolar traces reveals the populations of monomer and dimer states. Furthermore, we investigate the changes in these aspects as a function of buffer conditions and against the introduction of compounds that promote dimerization of the protein. These findings exemplify the capacity of EPR to contribute to questions across the PPI space, and the principles demonstrated here can be easily extrapolated toward other hot-topics within the biopharmaceutical industry, such as induced proximity and targeted protein degradation.
Austin Gamble Jarvi, Applications Manager, High Q Technologies