Site-directed spin labeling of membrane proteins.

EPR spectroscopy of site-directed spin labeled membrane proteins is at present a common and valuable biophysical tool to study structural details and conformational transitions under conditions relevant to function. EPR is considered a complementary approach to X-ray crystallography and NMR because it provides detailed information on (1) side chain dynamics with an exquisite sensitivity for flexible regions, (2) polarity and water accessibility profiles across the membrane bilayer, and (3) distances between two spin labeled side chains during protein functioning. Despite the drawback of requiring site-directed mutagenesis for each new piece of information to be collected, EPR can be applied to any complex membrane protein system, independently of its size. This chapter describes the state of the art in the application of site-directed spin labeling (SDSL) EPR to membrane proteins, with specific focus on the different types of information which can be obtained with continuous wave and pulsed techniques.