Site directed spin labeling to elucidating the mechanism of the cyanobacterial circadian clock.

Abstract

Electron Paramagnetic Resonance (EPR) is a spectroscopic technique that provides structural and dynamic information on unpaired spins and their surrounding environments. Introduction of exogenous spin labels via site directed spin labeling (SDSL) enables characterization of systems of interests lacking intrinsic unpaired spins. This chapter describes the use of SDSL in quantifying KaiB-KaiC binding in the cyanobacterial circadian clock (Kai Clock), exploiting the changes in mobility of the local environment around the spin label on KaiB-KaiC interactions. While the Kai system serves as our model system to demonstrate SDSL-EPR utility in quantifying protein-protein interactions, this technique is readily amenable to other systems of interest whenever specific protein-protein interactions need to be isolated. We first present a protocol for spin labeling KaiB. Then, we detail the sample preparation and acquisition processes to maximize signal-to-noise for downstream analysis. We close this chapter by highlighting recent advances in SDSL technology to incorporate spin labels into proteins of interest and in EPR technology to improve detection sensitivity that may allow greater flexibilities to the types of experiments possible.