Erythrosin B as a New Photoswitchable Spin Label for Light-Induced Pulsed EPR Dipolar Spectroscopy
Arnau Bertran,
Laura Morbiato,
Sara Aquilia,
Laura Gabbatore,
Marta De Zotti,
Christiane R. Timmel,
Marilena Di Valentin,
Alice M. Bowen
Affiliations
Arnau Bertran
Centre for Advanced Electron Spin Resonance and Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QR, UK
Laura Morbiato
Department of Chemical Sciences, University of Padova, 35131 Padova, Italy
Sara Aquilia
Department of Chemical Sciences, University of Padova, 35131 Padova, Italy
Laura Gabbatore
Department of Chemical Sciences, University of Padova, 35131 Padova, Italy
Marta De Zotti
Department of Chemical Sciences, University of Padova, 35131 Padova, Italy
Christiane R. Timmel
Centre for Advanced Electron Spin Resonance and Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QR, UK
Marilena Di Valentin
Department of Chemical Sciences, University of Padova, 35131 Padova, Italy
Alice M. Bowen
The National Research Facility for Electron Paramagnetic Resonance, Department of Chemistry and Photon Science Institute, The University of Manchester, Manchester M13 9PL, UK
We present a new photoswitchable spin label for light-induced pulsed electron paramagnetic resonance dipolar spectroscopy (LiPDS), the photoexcited triplet state of erythrosin B (EB), which is ideal for biological applications. With this label, we perform an in-depth study of the orientational effects in dipolar traces acquired using the refocused laser-induced magnetic dipole technique to obtain information on the distance and relative orientation between the EB and nitroxide labels in a rigid model peptide, in good agreement with density functional theory predictions. Additionally, we show that these orientational effects can be averaged to enable an orientation-independent analysis to determine the distance distribution. Furthermore, we demonstrate the feasibility of these experiments above liquid nitrogen temperatures, removing the need for expensive liquid helium or cryogen-free cryostats. The variety of choices in photoswitchable spin labels and the affordability of the experiments are critical for LiPDS to become a widespread methodology in structural biology.
