Orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies

Galazzo, Laura; Teucher, Markus; Bordignon, Enrica

doi:10.1016/bs.mie.2022.02.004

Cited by 15 publications

(9 citation statements)

References 138 publications

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“…The most often-used spin-labels are nitroxide radicals, with an electron spin of 1 / 2 and a nuclear spin of 1 (corresponds to 14 N). , The most widely used nitroxide spin-label is (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate (MTSL) (Figure A), which can be chemically attached to the thiol group of a cysteine residue. − This strategy usually requires generation of a mutant lacking all native cysteine residues together with the introduction of at least one cysteine residue via site-directed mutagenesis. The cysteine thiol groups specifically react with functional groups of the spin-label that create a covalent bond with the amino acid.…”

Section: Methodsmentioning

confidence: 99%

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

Hofmann

Ruthstein

2022

J. Phys. Chem. B

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In the last 20 years, the use of electron paramagnetic resonance (EPR) has made a pronounced and lasting impact in the field of structural biology. The advantage of EPR spectroscopy over other structural techniques is its ability to target even minor conformational changes in any biomolecule or macromolecular complex, independent of its size or complexity, or whether it is in solution or in the cell during a biological or chemical reaction. Here, we focus on the use of EPR spectroscopy to study transmembrane transport and transcription mechanisms. We discuss experimental and analytical concerns when referring to studies of two biological reaction mechanisms, namely, transfer of copper ions by the human copper transporter hCtr1 and the mechanism of action of the Escherichia coli copper-dependent transcription factor CueR. Last, we elaborate on future avenues in the field of EPR structural biology.

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Section: Methodsmentioning

confidence: 99%

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

Hofmann

Ruthstein

2022

J. Phys. Chem. B

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“…As a result, the interpretation of the spectrum is much simpler. We emphasize that the purpose of using such orthogonal labels is not merely to resolve their distinct spectral signatures 43 : rather, it is to simplify the form of the inter-label interaction itself for all label orientations.…”

Section: Numerical Simulationsmentioning

confidence: 99%

Detection of molecular transitions with nitrogen-vacancy centers and electron-spin labels

et al. 2022

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We present a protocol that detects molecular conformational changes with two nitroxide electron-spin labels and a nitrogen-vacancy (NV) center in diamond. More specifically, we demonstrate that the NV can detect energy shifts induced by the coupling between electron-spin labels. The protocol relies on the judicious application of microwave and radiofrequency pulses in a range of parameters that ensures stable nitroxide resonances. Furthermore, we demonstrate that our scheme is optimized by using nitroxides with distinct nitrogen isotopes. We develop a simple theoretical model that we combine with Bayesian inference techniques to demonstrate that our method enables the detection of conformational changes in ambient conditions including strong NV dephasing rates as a consequence of the diamond surface proximity and nitroxide thermalization mechanisms. Finally, we counter-intuitively show that with our method the small residual effect of random molecular tumbling becomes a resource that can be exploited to extract inter-label distances.

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“…NO and Gd can be also combined for DEER investigations using the so-called orthogonal spin labelling strategies (ortho-SL). [22,23] This approach is particularly advantageous to study systems where the presence of multiple paramagnetic centres with analogue EPR signatures can complicate the assignment of pairwise spin-spin distances. Since these spin labels show different EPR spectra, NOÀ Gd distances can be selectively probed by adjusting the experimental DEER setup in orthogonally labelled samples.…”

Section: Introductionmentioning

confidence: 99%

“…Other types of spin labels, such as Gd III ‐base tags (Gd, Figure 1B) [19,20] or trityl radicals, [21] have demonstrated to be excellent probes for DEER measurements. NO and Gd can be also combined for DEER investigations using the so‐called orthogonal spin labelling strategies ( ortho ‐SL) [22,23] . This approach is particularly advantageous to study systems where the presence of multiple paramagnetic centres with analogue EPR signatures can complicate the assignment of pairwise spin‐spin distances.…”

Section: Introductionmentioning

confidence: 99%

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw‐EPR and Nitroxide–Gd^III Distance Measurements

et al. 2023

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The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo-multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex. Here, we applied continuous-wave and pulse EPR spectroscopy using nitroxide-gadolinium orthogonal labelling strategies to investigate the hetero-oligomeric assembly of the minor pilins. Overall, our data are in line with the endopilus model even if they evidenced conformational flexibility and alternative orientations at local scale of specific regions of minor pilins. The integration of different labelling strategies and EPR experiments demonstrates the pertinence of this approach to investigate protein-protein interactions in such multiprotein heterocomplexes.

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Orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies

Cited by 15 publications

References 138 publications

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

Detection of molecular transitions with nitrogen-vacancy centers and electron-spin labels

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw‐EPR and Nitroxide–Gd^III Distance Measurements

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Orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies

Cited by 15 publications

References 138 publications

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

Detection of molecular transitions with nitrogen-vacancy centers and electron-spin labels

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw‐EPR and Nitroxide–GdIII Distance Measurements

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Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw‐EPR and Nitroxide–Gd^III Distance Measurements