Chapter 10. In-cell EPR

Abstract

Electron paramagnetic resonance (EPR) experiments on spin-labeled biomacromolecules yield information on the local conformation and dynamics of the labeling site and precise long-range distance constraints between pairs of spin labels. The technique is independent of the size of the studied macromolecule and essentially background-free in most environments. Consequently, EPR spectroscopy is well suited for the structural characterization of biomacromolecules directly in the cell, with double electron–electron resonance (DEER) being the most commonly employed technique for intracellular EPR distance measurements. In practice, a crucial point is the choice of the spin label, which should be stable in the reductive intracellular environment. While the most common approach involves transfection of the spin-labeled macromolecules into the studied cells, several strategies for in vivo labeling of endogenously expressed proteins have been introduced recently. With the ongoing development and optimization of experimental strategies and spectroscopic techniques, in-cell EPR is currently becoming more widely applicable to relevant biological questions, and is about to be established as a valuable tool for in-cell structural biology.