Orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies.

Different types of spin labels are currently available for structural studies of biomolecules both in vitro and in cells using Electron Paramagnetic Resonance (EPR) and pulse dipolar spectroscopy (PDS). Each type of label has its own advantages and disadvantages, that will be addressed in this chapter. The spectroscopically distinct properties of the labels have fostered new applications of PDS aimed to simultaneously extract multiple inter-label distances on the same sample. In fact, combining different labels and choosing the optimal strategy to address their inter-label distances can increase the information content per sample, and this is pivotal to better characterize complex multi-component biomolecular systems. In this review, we provide a brief background of the spectroscopic properties of the four most common orthogonal spin labels for PDS measurements and focus on the various methods at disposal to extract homo- and hetero-label distances in proteins. We also devote a section to possible artifacts arising from channel crosstalk and provide few examples of applications in structural biology.