CW-EPR Spectroscopy and Site-Directed Spin Labeling to Study the Structural Dynamics of Ion Channels

Abstract

Continuous-wave electron paramagnetic resonance spectroscopy (CW-EPR) and site-directed spin labeling (SDSL) are proven experimental approaches to assess the structural dynamics of proteins in general (Hubbell et al., Curr Opin Struct Biol 8(5):649–656, 1998; Kazmier et al., Curr Opin Struct Biol 45:100–108, 2016; Perozo et al., Science 285(5424):73–78, 1999). These techniques have been particularly effective assessing the structure of integral membrane proteins embedded in a lipid bilayer (Cortes et al., J Gen Physiol 117(2):165–180, 2001; Cuello et al., Science 306(5695):491–495, 2004; Dalmas et al., Structure 18(7):868–878, 2010; Li et al., Proc Natl Acad Sci U S A 112(44):E5926–5935, 2015; Perozo et al., J Gen Physiol 118(2):193–206, 2001), as well as determining the conformational changes associated with their biological function (Kazmier et al., Curr Opin Struct Biol 45:100–108, 2016; Perozo et al., Science 285(5424):73–78, 1999; Arrigoni et al., Cell 164(5):922–936, 2016; Dalmas et al., Nat Commun 5:3590, 2014; Dong et al., Science 308(5724):1023–1028, 2005; Farrens et al., Science 274(5288):768–770, 1996; Perozo et al., Nat Struct Biol 5(6):459–469, 1998; Perozo et al., Nature 418(6901):942–948, 2002). In this chapter, we described a practical guide for the spin-labeling, liposome reconstitution, and CW-EPR measurements of the prototypical bacterial K⁺ channel, KcsA.