Tracking Copper sensing operon Repressor (CsoR) oligomerization in solution using Electron Paramagnetic Resonance spectroscopy.

Protein oligomerization is a fundamental biological process that plays a pivotal role in regulating numerous cellular functions. In this study, we investigated the oligomeric state of copper-sensing operon repressor (CsoR), a transcriptional repressor bound to the cso operon in Mycobacterium tuberculosis. Upon copper binding, CsoR dissociates from the promoter region, permitting transcription initiation. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate the oligomeric state of CsoR as a function of DNA binding. The data indicated that CsoR exists predominantly as a tetramer in equilibrium with some dimer population, independent of DNA binding. Room temperature continuous wave EPR measurements suggested the presence of two components, where the immobile component was related to spin-labeled CsoR dimers within a tetrameric assembly, and the mobile component was associated with the dimeric assembly. Relaxation measurements were used to verify this assignment, and double electron electron resonance experiments were used to characterize the oligomeric state. Using this approach, we showed that although the equilibrium between tetramers and dimers is preserved as a function of DNA binding, the exchange between the different protein units is affected by the presence of DNA, which likely contributes to the transcription regulation.