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

Bizet, Maxime and Balázsi, Áron and Byrne, Deborah and Gerbaud, Guillaume and Kálai, Tamás and Martinho, Marlène (2026) Incorporating Noncanonical Amino Acids for Protein Structure Analysis: Site-Directed Spin Labeling CW and Pulsed EPR Approaches. CHEMICAL REVIEWS. ISSN 0009-2665 (In Press)

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Abstract

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.

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