Nitroxide spin, non-canonical amino acids and SDSL-EPR spectroscopy:a winning combination for protein dynamics studies

Bizet, Maxime and Balázsi, Áron and Maignet-Magard, Colette and Dorlet, Pierre and Kálai, Tamás and Martinho, Marlène and Etienne, Emilien and Gerbaud, Guillaume and Gilles, Truan and Byrne, Deborah (2026) Nitroxide spin, non-canonical amino acids and SDSL-EPR spectroscopy:a winning combination for protein dynamics studies. EUROPEAN BIOPHYSICS JOURNAL. ISSN 0175-7571 (print); 1432-1017 (online)

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Abstract

Site-Directed Spin Labelling combined with Electron Paramagnetic Resonance (SDSL-EPR) is a powerful technique for studying protein dynamics at the molecular level. This method involves introducing a paramagnetic label, typically at a cysteine residue at a selected site. Continuous wave (cw) EPR allows studying protein dynamics in the nano-second time scale, while using Double Electron Electron Resonance (DEER), nanometre-scale (2–6 nm) distance measurements can be obtained by detecting dipolar interaction between two paramagnetic labels. Here, we describe the application of the SDSL-EPR approach to the soluble form of the Homo sapiens flavoprotein cytochrome P450 reductase, utilizing its stabilized semi-quinone state (FMNH•) as an intrinsic paramagnetic centre. Additionally, the non-canonical amino acid (ncaa) pAzPhe (para-azidophenylalanine) was site-specifically incorporated into the FAD (Flavin Adenine Dinucleotide) and FMN (Flavin MonoNucleotide) domains, and labelled with the recently developed nitroxide probes 14/15N-HO-5223. DEER measurements were performed between the FMNH• centre and the nitroxide label, providing structural insights into the protein. This protocol details the synthesis of the 14/15N-HO-5223 label, the incorporation of pAzPhe at the two specific positions, site-specific labelling and EPR measurements, offering a comprehensive workflow/protocol for applying SDSL-EPR to flavoproteins and beyond.

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