Electron Paramagnetic Resonance in Structural Biology

Abstract

This chapter presents the application of electron paramagnetic resonance (EPR) spectroscopy to exploring the structure and dynamics of biomolecules, primarily proteins. Emphasis is given to pulsed dipolar EPR (PD-EPR), which provides distance distributions, typically in the range of 1.5–8 nm. Such measurements, usually carried out at low temperatures on frozen samples, are well established in solution and have been demonstrated as effective for in-cell studies. The chapter begins with a brief description of continuous wave EPR, followed by the most commonly used PD-EPR techniques, along with the associated data analysis methodology. Since most biomolecules are diamagnetic, site-directed spin labeling plays an essential role in EPR applications. The various spin labels currently available are discussed next. This is followed by a description of approaches used to model the rotational freedom of the spin label upon binding to specific sites in known structures and a prediction of the ensuing distance distribution. Then the use of distance distributions as constraints in structural derivation is briefly presented. The chapter concludes by showing some examples of the concurrent use of EPR techniques with other biophysical methods, such as NMR, SAXS, smFRET, and cryo-EM, highlighting actual biological applications, rather than model proteins often used for new method development.