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

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Title: Nitroxide spin, non-canonical amino acids and SDSL-EPR spectroscopy: a winning combination for protein dynamics studies.
Authors: Bizet, Maxime1 (AUTHOR), Balázsi, Áron2 (AUTHOR), Maignet-Magard, Colette1 (AUTHOR), Dorlet, Pierre1 (AUTHOR), Etienne, Emilien1 (AUTHOR), Gerbaud, Guillaume1 (AUTHOR), Truan, Gilles3 (AUTHOR), Byrne, Deborah4 (AUTHOR) byrne@imm.cnrs.fr, Kálai, Tamás2,5 (AUTHOR) tamas.kalai@aok.pte.hu, Martinho, Marlène1 (AUTHOR) mmartinho@imm.cnrs.fr
Source: European Biophysics Journal. Jun2026, Vol. 55 Issue 3, p347-365. 19p.
Subjects: Electron paramagnetic resonance, Nitroxides, Spin labels, Amino acids, Cytochrome P-450, Flavoproteins, Protein conformation
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. [ABSTRACT FROM AUTHOR]
Copyright of European Biophysics Journal is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
Database: Engineering Source
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DbLabel: Engineering Source
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  Data: Nitroxide spin, non-canonical amino acids and SDSL-EPR spectroscopy: a winning combination for protein dynamics studies.
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  Data: <searchLink fieldCode="AR" term="%22Bizet%2C+Maxime%22">Bizet, Maxime</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Balázsi%2C+Áron%22">Balázsi, Áron</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Maignet-Magard%2C+Colette%22">Maignet-Magard, Colette</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Dorlet%2C+Pierre%22">Dorlet, Pierre</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Etienne%2C+Emilien%22">Etienne, Emilien</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Gerbaud%2C+Guillaume%22">Gerbaud, Guillaume</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Truan%2C+Gilles%22">Truan, Gilles</searchLink><relatesTo>3</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Byrne%2C+Deborah%22">Byrne, Deborah</searchLink><relatesTo>4</relatesTo> (AUTHOR)<i> byrne@imm.cnrs.fr</i><br /><searchLink fieldCode="AR" term="%22Kálai%2C+Tamás%22">Kálai, Tamás</searchLink><relatesTo>2,5</relatesTo> (AUTHOR)<i> tamas.kalai@aok.pte.hu</i><br /><searchLink fieldCode="AR" term="%22Martinho%2C+Marlène%22">Martinho, Marlène</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> mmartinho@imm.cnrs.fr</i>
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  Data: <searchLink fieldCode="JN" term="%22European+Biophysics+Journal%22">European Biophysics Journal</searchLink>. Jun2026, Vol. 55 Issue 3, p347-365. 19p.
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  Data: <searchLink fieldCode="DE" term="%22Electron+paramagnetic+resonance%22">Electron paramagnetic resonance</searchLink><br /><searchLink fieldCode="DE" term="%22Nitroxides%22">Nitroxides</searchLink><br /><searchLink fieldCode="DE" term="%22Spin+labels%22">Spin labels</searchLink><br /><searchLink fieldCode="DE" term="%22Amino+acids%22">Amino acids</searchLink><br /><searchLink fieldCode="DE" term="%22Cytochrome+P-450%22">Cytochrome P-450</searchLink><br /><searchLink fieldCode="DE" term="%22Flavoproteins%22">Flavoproteins</searchLink><br /><searchLink fieldCode="DE" term="%22Protein+conformation%22">Protein conformation</searchLink>
– Name: Abstract
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  Data: 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. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
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  Data: <i>Copyright of European Biophysics Journal is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1007/s00249-025-01806-3
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      – Code: eng
        Text: English
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        PageCount: 19
        StartPage: 347
    Subjects:
      – SubjectFull: Electron paramagnetic resonance
        Type: general
      – SubjectFull: Nitroxides
        Type: general
      – SubjectFull: Spin labels
        Type: general
      – SubjectFull: Amino acids
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      – SubjectFull: Cytochrome P-450
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      – SubjectFull: Flavoproteins
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      – SubjectFull: Protein conformation
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      – TitleFull: Nitroxide spin, non-canonical amino acids and SDSL-EPR spectroscopy: a winning combination for protein dynamics studies.
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              Text: Jun2026
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