Incorporating Noncanonical Amino Acids for Protein Structure Analysis: Site-Directed Spin Labeling CW and Pulsed EPR Approaches

Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy (SDSL−EPR) has emerged as a powerful and highly adaptable platform probing protein structure and dynamics with residue-level precision. In its classical strategy, a stable paramagnetic label, most commonly a nitroxide radical, but not only, is covalently introduced at a predefined position, typically through an engineered cysteine 11 residue. Continuous-wave (cw) EPR spectroscopy provides sensitivity to protein dynamics across a broad range of time scales (and techniques), from backbone motions (ps-ns) to protein complex assembly and conformational transitions (ms-s) or characterization of protein conformational exchange (μs-ms). Pulsed EPR methodologies, particularly double electron−electron resonance (DEER), provide access to long-range distance distributions in the nanometer regime (ca. 2−8 nm) through the measurement of dipolar interactions between pairs of spin labels. Recent developments in the genetic encoding of noncanonical amino acids (ncAAs) have substantially expanded the scope of SDSL−EPR by overcoming the intrinsic limitations associated with cysteine-based labeling. In this review, we provide a comprehensive overview of ncAA-based spin-labeling strategies for SDSL−EPR, discuss the repertoire of reported spin labels and their synthetic routes, and highlight representative applications in structural investigations of proteins in vitro as well as in cellular environments.