Genetically Encoded Spin Labels for In Vitro and In-Cell EPR Studies of Native Proteins

Abstract Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to study the structure, dynamics, and interactions of proteins. The genetic encoding of the noncanonical amino acid spin-labeled lysine 1 (SLK-1) eliminates the need for any chemical labeling steps in SDSL–EPR studies and enables the investigation of native, endogenous proteins with minimal structural perturbation, and without the need to create unique reactive sites for chemical labeling. We report detailed experimental procedures for the efficient synthesis of SLK-1, the expression and purification of SLK-1-containing proteins under conditions that ensure maximal integrity of the nitroxide radical moiety, and procedures for intramolecular EPR distance measurements in proteins by double electron–electron resonance.