Guidelines for the Simulations of Nitroxide X-Band cw EPR Spectra from Site-Directed Spin Labeling Experiments Using S imLabel

Emilien Etienne; Annalisa Pierro; Ketty C Tamburrini; Alessio Bonucci; Elisabetta Mileo; Marlène Martinho; Valérie Belle

doi:10.3390/molecules28031348

Guidelines for the Simulations of Nitroxide X-Band cw EPR Spectra from Site-Directed Spin Labeling Experiments Using S imLabel

Molecules. 2023 Jan 31;28(3):1348. doi: 10.3390/molecules28031348.

Authors

Emilien Etienne¹, Annalisa Pierro^{1

2}, Ketty C Tamburrini^{1

3

4}, Alessio Bonucci¹, Elisabetta Mileo¹, Marlène Martinho¹, Valérie Belle¹

Affiliations

¹ Aix Marseille University, CNRS (Centre National de la Recherche Scientifique), BIP (Bioénergétique et Ingénierie des Protéines), IMM (Institut de Microbiologie de la Méditerranée), 13009 Marseille, France.
² Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.
³ Aix Marseille University, CNRS (Centre National de la Recherche Scientifique), AFMB (Architecture et Fonction des Macromolécules Biologiques), 13009 Marseille, France.
⁴ Aix Marseille University, INRAE (Institut National de Recherche Pour L'agriculture, L'alimentation et L'Environnement), BBF (Biodiversité et Biotechnologies Fongiques), 13009 Marseille, France.

Abstract

Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the local level, the dynamics of structural transitions in proteins. Here, we consider SDSL-EPR based on the selective grafting of a nitroxide on the protein under study, followed by X-band cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give a reliable interpretation on biological system dynamics, a numerical simulation of the spectra is required. However, regardless of the numerical tool chosen to perform such simulations, the number of parameters is often too high to provide unambiguous results. In this study, we have chosen SimLabel to perform such simulations. SimLabel is a graphical user interface (GUI) of Matlab, using some functions of Easyspin. An exhaustive review of the parameters used in this GUI has enabled to define the adjustable parameters during the simulation fitting and to fix the others prior to the simulation fitting. Among them, some are set once and for all (g_y, g_z) and others are determined (A_z, g_x) thanks to a supplementary X-band spectrum recorded on a frozen solution. Finally, we propose guidelines to perform the simulation of X-band cw-EPR spectra of nitroxide labeled proteins at room temperature, with no need of uncommon higher frequency spectrometry and with the minimal number of variable parameters.

Keywords: EPR; ESR; EasySpin; SimLabel; nitroxide; simulation; site-directed spin labeling.

MeSH terms

Electron Spin Resonance Spectroscopy / methods
Nitrogen Oxides* / chemistry
Proteins* / chemistry
Spin Labels

Substances

Spin Labels
nitroxyl
Nitrogen Oxides
Proteins

Grants and funding

The authors are grateful to the EPR facilities available at the French EPR network (Infranalytics, FR2054), the Aix-Marseille EPR center and the MOSBRI project, that has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement N° 101004806. We acknowledge financial support from the “Agence Nationale de la Recherche” (ANR-18-CE11-0007-01) and from the “Conseil Régional Région Sud” (A.P. PhD Fellowship EJD-2018-2021). The project leading to this publication has also received funding from the A*Midex Foundation of Aix-Marseille University, funded by socio-economic partners.