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In situ observation of conformational dynamics and protein ligand–substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy

Nature Protocols volume 14, pages 2344–2369 (2019)Cite this article

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Abstract

Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5–7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4–5 d. The described protocol provides a general strategy for observing protein ligand–substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.

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Fig. 1: Pulse sequences for DEER/PELDOR spectroscopy.
Fig. 2: In situ PELDOR in native OM.
Fig. 3: Schematic view of the cell envelope of Gram-negative bacteria.
Fig. 4: In situ PELDOR in E. coli.
Fig. 5: In situ MTSL labeling of BtuB in E. coli.

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Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. McHaourab, H. S., Steed, P. R. & Kazmier, K. Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy. Structure 19, 1549–1561 (2011).

    Article  CAS  Google Scholar 

  2. Laganowsky, A. et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014).

    Article  CAS  Google Scholar 

  3. Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).

    Article  CAS  Google Scholar 

  4. Jeschke, G. Conformational dynamics and distribution of nitroxide spin labels. Prog. Nucl. Mag. Res. Sp. 72, 42–60 (2013).

    Article  CAS  Google Scholar 

  5. Hubbell, W. L. & Altenbach, C. Investigation of structure and dynamics in membrane proteins using site-directed spin labeling. Curr. Opin. Struct. Biol. 4, 566–573 (1994).

    Article  CAS  Google Scholar 

  6. Fleissner, M. R. et al. Site-directed spin labeling of a genetically encoded unnatural amino acid. Proc. Natl. Acad. Sci. USA 106, 21637–21642 (2009).

    Article  CAS  Google Scholar 

  7. Schmidt, M. J., Borbas, J., Drescher, M. & Summerer, D. A genetically encoded spin label for electron paramagnetic resonance distance measurements. J. Am. Chem. Soc. 136, 1238–1241 (2014).

    Article  CAS  Google Scholar 

  8. Abdelkader, E. H. et al. Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-L-phenylalanine. Chem. Commun. 51, 15898–15901 (2015).

    Article  CAS  Google Scholar 

  9. Milov, A. D., Ponomarev, A. B. & Tsvetkov, Y. D. Electron-electron double resonance in electron spin echo: model biradical systems and the sensitized photolysis of decalin. Chem. Phys. Lett. 110, 67–72 (1984).

    Article  CAS  Google Scholar 

  10. Martin, R. E. et al. Determination of end-to-end distances in a series of TEMPO diradicals of up to 2.8 nm length with a new four-pulse double electron electron resonance experiment. Angew. Chem. Int. Ed. 37, 2833–2837 (1998).

    Article  Google Scholar 

  11. Schiemann, O. et al. Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances. Nat. Protoc. 2, 904–923 (2007).

    Article  CAS  Google Scholar 

  12. Igarashi, R. et al. Distance determination in proteins inside Xenopus laevis oocytes by double electron-electron resonance experiments. J. Am. Chem. Soc. 132, 8228–8229 (2010).

    Article  CAS  Google Scholar 

  13. Jeschke, G. DEER distance measurements on proteins. Ann. Rev. Phys. Chem. 63, 419–446 (2012).

    Article  CAS  Google Scholar 

  14. Martorana, A. et al. Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labeling. J. Am. Chem. Soc. 136, 13458–13465 (2014).

    Article  CAS  Google Scholar 

  15. Azarkh, M. et al. Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment. Nat. Protoc. 8, 131–147 (2013).

    Article  CAS  Google Scholar 

  16. Schmidt, T., Walti, M. A., Baber, J. L., Hustedt, E. J. & Clore, G. M. Long distance measurements up to 160 A in the GroEL tetradecamer using Q-band DEER EPR spectroscopy. Angew. Chem. Int. Ed. 55, 15905–15909 (2016).

    Article  CAS  Google Scholar 

  17. Claxton, D. P., Kazmier, K., Mishra, S. & McHaourab, H. S. Navigating membrane protein structure, dynamics, and energy landscapes using spin labeling and EPR spectroscopy. Methods Enzymol. 564, 349–387 (2015).

    Article  CAS  Google Scholar 

  18. Barth, K. et al. Conformational coupling and trans-inhibition in the human antigen transporter ortholog TmrAB resolved with dipolar EPR spectroscopy. J. Am. Chem. Soc. 140, 4527–4533 (2018).

    Article  CAS  Google Scholar 

  19. Bordignon, E. & Bleicken, S. New limits of sensitivity of site-directed spin labeling electron paramagnetic resonance for membrane proteins. Biochim. Biophys. Acta 1860, 841–853 (2018).

    Article  CAS  Google Scholar 

  20. Dastvan, R., Fischer, A. W., Mishra, S., Meiler, J. & McHaourab, H. S. Protonation-dependent conformational dynamics of the multidrug transporter EmrE. Proc. Natl. Acad. Sci. USA 113, 1220–1225 (2016).

    Article  CAS  Google Scholar 

  21. Polyhach, Y., Bordignon, E. & Jeschke, G. Rotamer libraries of spin labelled cysteines for protein studies. Phys. Chem. Chem. Phys. 13, 2356–2366 (2011).

    Article  CAS  Google Scholar 

  22. Jeschke, G. & Polyhach, Y. Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance. Phys. Chem. Chem. Phys. 9, 1895–1910 (2007).

    Article  CAS  Google Scholar 

  23. Kattnig, D. R., Reichenwallner, J. & Hinderberger, D. Modeling excluded volume effects for the faithful description of the background signal in double electron-electron resonance. J. Phys. Chem. B 117, 16542–16557 (2013).

    Article  CAS  Google Scholar 

  24. Jeschke, G., Panek, G., Godt, A., Bender, A. & Paulsen, H. Data analysis procedures for pulse ELDOR measurements of broad distance distributions. Appl. Magn. Reson. 26, 223–244 (2004).

    Article  CAS  Google Scholar 

  25. Chiang, Y. W., Borbat, P. P. & Freed, J. H. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. J. Magn. Reson. 172, 279–295 (2005).

    Article  CAS  Google Scholar 

  26. Jeschke, G. et al. DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data. Appl. Magn. Reson. 30, 473–498 (2006).

    Article  CAS  Google Scholar 

  27. Edwards, T. H. & Stoll, S. A Bayesian approach to quantifying uncertainty from experimental noise in DEER spectroscopy. J. Magn. Reson. 270, 87–97 (2016).

    Article  CAS  Google Scholar 

  28. Stein, R. A., Beth, A. H. & Hustedt, E. J. A straightforward approach to the analysis of double electron-electron resonance data. Methods Enzymol. 563, 531–567 (2015).

    Article  CAS  Google Scholar 

  29. Borbat, P. P., Georgieva, E. R. & Freed, J. H. Improved sensitivity for long-distance measurements in biomolecules: five-pulse double electron-electron resonance. J. Phys. Chem. Lett. 4, 170–175 (2013).

    Article  CAS  Google Scholar 

  30. Spindler, P. E. et al. Carr-purcell pulsed electron double resonance with shaped inversion pulses. J. Phys. Chem. Lett. 6, 4331–4335 (2015).

    Article  CAS  Google Scholar 

  31. Saxena, S. & Freed, J. H. Theory of double quantum two-dimensional electron spin resonance with application to distance measurements. J. Chem. Phys. 107, 1317–1340 (1997).

    Article  CAS  Google Scholar 

  32. Jeschke, G., Pannier, M., Godt, A. & Spiess, H. W. Dipolar spectroscopy and spin alignment in electron paramagnetic resonance. Chem. Phys. Lett. 331, 243–252 (2000).

    Article  CAS  Google Scholar 

  33. Milikisyants, S., Scarpelli, F., Finiguerra, M. G., Ubbink, M. & Huber, M. A pulsed EPR method to determine distances between paramagnetic centers with strong spectral anisotropy and radicals: the dead-time free RIDME sequence. J. Magn. Reson. 201, 48–56 (2009).

    Article  CAS  Google Scholar 

  34. Razzaghi, S. et al. RIDME spectroscopy with Gd(III) centers. J. Phys. Chem. Lett. 5, 3970–3975 (2014).

    Article  CAS  Google Scholar 

  35. Lawless, M. J. et al. Analysis of nitroxide-based distance measurements in cell extracts and in cells by pulsed ESR spectroscopy. ChemPhysChem 18, 1653–1660 (2017).

    Article  CAS  Google Scholar 

  36. Dunkel, S., Pulagam, L. P., Steinhoff, H. J. & Klare, J. P. In vivo EPR on spin labeled colicin A reveals an oligomeric assembly of the pore-forming domain in E. coli membranes. Phys. Chem. Chem. Phys. 17, 4875–4878 (2015).

    Article  CAS  Google Scholar 

  37. Jagtap, A. P. et al. Sterically shielded spin labels for in-cell EPR spectroscopy: analysis of stability in reducing environment. Free Radic. Res. 49, 78–85 (2015).

    Article  CAS  Google Scholar 

  38. Karthikeyan, G. et al. A bioresistant nitroxide spin label for in-cell EPR spectroscopy: in vitro and in oocytes protein structural dynamics studies. Angew. Chem. Int. Ed. 57, 1366–1370 (2018).

    Article  CAS  Google Scholar 

  39. Qi, M., Gross, A., Jeschke, G., Godt, A. & Drescher, M. Gd(III)-PyMTA label is suitable for in-cell EPR. J. Am. Chem. Soc. 136, 15366–15378 (2014).

    Article  CAS  Google Scholar 

  40. Theillet, F. X. et al. Structural disorder of monomeric alpha-synuclein persists in mammalian cells. Nature 530, 45–50 (2016).

    Article  CAS  Google Scholar 

  41. Yang, Y. et al. A reactive, rigid Gd-III labeling tag for in-cell EPR distance measurements in proteins. Angew. Chem. Int. Ed. 56, 2914–2918 (2017).

    Article  CAS  Google Scholar 

  42. Prokopiou, G. et al. Small Gd(III) Tags for Gd(III)-Gd(III) distance measurements in proteins by EPR spectroscopy. Inorg. Chem. 57, 5048–5059 (2018).

    Article  CAS  Google Scholar 

  43. Reginsson, G. W., Kunjir, N. C., Sigurdsson, S. T. & Schiemann, O. Trityl radicals: spin labels for nanometer-distance measurements. Chemistry 18, 13580–13584 (2012).

    Article  CAS  Google Scholar 

  44. Joseph, B. et al. Selective high-resolution detection of membrane protein-ligand interaction in native membranes using trityl-nitroxide PELDOR. Angew. Chem. Int. Ed. 55, 11538–11542 (2016).

    Article  CAS  Google Scholar 

  45. Jassoy, J. J. et al. Versatile trityl spin labels for nanometer distance measurements on biomolecules in vitro and within cells. Angew. Chem. Int. Ed. 56, 177–181 (2017).

    Article  CAS  Google Scholar 

  46. Jiang, X. et al. Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria. Science 276, 1261–1264 (1997).

    Article  CAS  Google Scholar 

  47. Joseph, B. et al. Distance measurement on an endogenous membrane transporter in E. coli cells and native membranes using EPR spectroscopy. Angew. Chem. Int. Ed. Engl. 54, 6196–6199 (2015).

    Article  CAS  Google Scholar 

  48. Joseph, B., Sikora, A. & Cafiso, D. S. Ligand induced conformational changes of a membrane transporter in E. coli cells observed with DEER/PELDOR. J. Am. Chem. Soc. 138, 1844–1847 (2016).

    Article  CAS  Google Scholar 

  49. Sikora, A., Joseph, B., Matson, M., Staley, J. R. & Cafiso, D. S. Allosteric signaling is bidirectional in an outer-membrane transport protein. Biophys. J. 111, 1908–1918 (2016).

    Article  CAS  Google Scholar 

  50. Fairman, J. W., Noinaj, N. & Buchanan, S. K. The structural biology of beta-barrel membrane proteins: a summary of recent reports. Curr. Opin. Struct. Biol. 21, 523–531 (2011).

    Article  CAS  Google Scholar 

  51. Gessmann, D. et al. Outer membrane beta-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA. Proc. Natl. Acad. Sci. USA 111, 5878–5883 (2014).

    Article  CAS  Google Scholar 

  52. Schiffrin, B. et al. Effects of periplasmic chaperones and membrane thickness on BamA-catalyzed outer-membrane protein folding. J. Mol. Biol. 429, 3776–3792 (2017).

    Article  CAS  Google Scholar 

  53. Rassam, P. et al. Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria. Nature 523, 333–336 (2015).

    Article  CAS  Google Scholar 

  54. Iadanza, M. G. et al. Lateral opening in the intact beta-barrel assembly machinery captured by cryo-EM. Nat. Commun. 7, 12865 (2016).

    Article  CAS  Google Scholar 

  55. Gu, Y. et al. Structural basis of outer membrane protein insertion by the BAM complex. Nature 531, 64–69 (2016).

    Article  CAS  Google Scholar 

  56. Okuda, S., Sherman, D. J., Silhavy, T. J., Ruiz, N. & Kahne, D. Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model. Nat. Rev. Microbiol. 14, 337–345 (2016).

    Article  CAS  Google Scholar 

  57. Robert, V. et al. Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif. PLoS Biol. 4, 1984–1995 (2006).

    Article  CAS  Google Scholar 

  58. Pinto, C. et al. Studying assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy. J. Struct. Biol. 206, 1–11 (2019).

    Article  CAS  Google Scholar 

  59. Wagner, J., Schaffer, M. & Fernandez-Busnadiego, R. Cryo-electron tomography-the cell biology that came in from the cold. FEBS Lett. 591, 2520–2533 (2017).

    Article  CAS  Google Scholar 

  60. Dimura, M. et al. Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems. Curr. Opin. Struct. Biol. 40, 163–185 (2016).

    Article  CAS  Google Scholar 

  61. Sustarsic, M. & Kapanidis, A. N. Taking the ruler to the jungle: single-molecule FRET for understanding biomolecular structure and dynamics in live cells. Curr. Opin. Struct. Biol. 34, 52–59 (2015).

    Article  CAS  Google Scholar 

  62. Fuller-Schaefer, C. A. & Kadner, R. J. Multiple extracellular loops contribute to substrate binding and transport by the Escherichia coli cobalamin transporter BtuB. J. Bacteriol. 187, 1732–1739 (2005).

    Article  CAS  Google Scholar 

  63. Sarver, J. L., Zhang, M., Liu, L. S., Nyenhuis, D. & Cafiso, D. S. A dynamic protein-protein coupling between the TonB-dependent transporter FhuA and TonB. Biochemistry 57, 1045–1053 (2018).

    Article  CAS  Google Scholar 

  64. Filip, C., Fletcher, G., Wulff, J. L. & Earhart, C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol. 115, 717–722 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Georgieva, E. R. et al. Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): a study of doubly-spin-labeled T4 lysozyme. J. Magn. Reson. 216, 69–77 (2012).

    Article  CAS  Google Scholar 

  66. Hagelueken, G., Ward, R., Naismith, J. H. & Schiemann, O. MtsslWizard: in silico spin-labeling and generation of distance distributions in PyMOL. Appl. Magn. Reson. 42, 377–391 (2012).

    Article  CAS  Google Scholar 

  67. Hatmal, M. M. et al. Computer modeling of nitroxide spin labels on proteins. Biopolymers 97, 35–44 (2012).

    Article  CAS  Google Scholar 

  68. Cadieux, N. & Kadner, R. J. Site-directed disulfide bonding reveals an interaction site between energy-coupling protein TonB and BtuB, the outer membrane cobalamin transporter. Proc. Natl. Acad. Sci. USA 96, 10673–10678 (1999).

    Article  CAS  Google Scholar 

  69. Peters, J. E., Thate, T. E. & Craig, N. L. Definition of the Escherichia coli MC4100 genome by use of a DNA array. J. Bacteriol. 185, 2017–2021 (2003).

    Article  CAS  Google Scholar 

  70. Baker, L. A., Daniels, M., van der Cruijsen, E. A., Folkers, G. E. & Baldus, M. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling. J. Biomol. NMR 62, 199–208 (2015).

    Article  CAS  Google Scholar 

  71. Polyhach, Y. et al. High sensitivity and versatility of the DEER experiment on nitroxide radical pairs at Q-band frequencies. Phys. Chem. Chem. Phys. 14, 10762–10773 (2012).

    Article  CAS  Google Scholar 

  72. Teucher, M. & Bordignon, E. Improved signal fidelity in 4-pulse DEER with Gaussian pulses. J. Magn. Reson. 296, 103–111 (2018).

    Article  CAS  Google Scholar 

  73. Tait, C. E. & Stoll, S. Coherent pump pulses in Double Electron Electron Resonance spectroscopy. Phys. Chem. Chem. Phys. 18, 18470–18485 (2016).

    Article  CAS  Google Scholar 

  74. Bode, B. E. et al. Counting the monomers in nanometer-sized oligomers by pulsed electron-electron double resonance. J. Am. Chem. Soc. 129, 6736–6745 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the Marie-Curie COFUND Postdoctoral program (PCOFUND-GA-2011-291776, GO-IN), the Adolf-Messer Foundation (B.J.), the Deutsche Forschungsgemeinschaft (SFB807 to B.J. and T.F.P.), and the National Institutes of Health, NIGMS (GM035215, D.S.C.) for financial support.

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Authors and Affiliations

  1. Institute of Biophysics, Department of Physics, University of Frankfurt, Frankfurt am Main, Germany

    Benesh Joseph

  2. Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, University of Frankfurt, Frankfurt am Main, Germany

    Benesh Joseph, Eva A. Jaumann, Katja Barth & Thomas F. Prisner

  3. Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, VA, USA

    Arthur Sikora & David S. Cafiso

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  1. Benesh Joseph
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  2. Eva A. Jaumann
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  3. Arthur Sikora
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Contributions

B.J. conceived the idea and initiated the project on in situ DEER/PELDOR of OMPs in E. coli and isolated OMs and further developed it in collaboration with D.S.C. in the laboratory of T.F.P. A.S. participated in mutagenesis and spin labeling at the beginning of the project. B.J. and K.B. synthesized TEMPO-HOCbl. B.J. performed all the PELDOR experiments discussed in the main text. B.J and E.A.J. performed further optimizations for MTSL labeling and wrote the manuscript with input from other co-authors.

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Correspondence to Benesh Joseph.

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Key references using this protocol

Joseph, B. et al. Angew. Chem. Int. Ed. Engl. 54, 6196–6199 (2015): https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201501086

Joseph, B., Sikora, A. & Cafiso, D. S. J. Am. Chem. Soc.138, 1844–1847 (2016): https://pubs.acs.org/doi/abs/10.1021/jacs.5b13382

Joseph, B. et al. Angew. Chem. Int. Ed. Engl. 55, 11538–11542 (2016): https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201606335

Sikora, A., Joseph, B., Matson, M., Staley, J. R. & Cafiso, D. S. Biophys. J.111, 1908–1918 (2016): https://www.cell.com/biophysj/fulltext/S0006-3495(16)30872-4

Integrated supplementary information

Supplementary Fig. 1 Observing conformational changes in E. coli.

(a) Conformational changes of the second loop was monitored with respect to the seventh loop in the absence (apo) or presence of the ligands (Ca2+ and cyanocobalamin, CN–cobalamin). The spin labeled positions are highlighted on the apo-BtuB structure (1NQE). The conformation of the loops as observed in the BtuB–Ca2+ (in blue, PDB 1NQG) and BtuB–CN–cobalamin+Ca2+ (in yellow, PDB 1NQH) structures are overlaid. The second loop is not resolved in the apo crystal structure. (b, d) Background corrected Q-band PELDOR data for the 188R1–399R1 mutant in E. coli (red) or OM (blue). (c, e) The corresponding distance distributions obtained with Tikhonov regularization. In agreement with the crystal structures, the second loop exhibits large conformational flexibility in the apo-state and binding of the ligands reduce the flexibility. Adapted with permission from ref. 48. Joseph, B., Sikora, A. & Cafiso, D. S. Ligand induced conformational changes of a membrane transporter in E. coli cells observed with DEER/PELDOR. J. Am. Chem. Soc. 138, 1844-1847. Copyright (2016) American Chemical Society.

Supplementary Fig. 2 In situ spin labeling and PELDOR in native OM.

Spin labeling or ligand binding is possible at both sides of the membrane. (a) MTSL labeling of BtuB T188C or the Cys-less (WT) protein in the cell envelope (IM+OM), which produced a rather similar spectrum. (b) MTSL labeling after removal of the IM, which gave a much larger signal for the T188C mutant as compared to the WT. (c) Background corrected Q-band PELDOR data between V10R1 and TEMPO-labelled cyanocobalamin (TEMPO-CNCbl). (d) The corresponding distance distribution obtained with Tikhonov regularization. Adapted with permission from ref. 47, Wiley.

Supplementary Fig. 3 RP-HPLC of the TEMPO-HOCbl preparation with detection at 316 nm.

The major peak is highlighted and an overlay of its UV-Vis spectra with the same for cyanocobalamin is shown as an inset on the left. This peak revealed a strong EPR signal. The shoulder on the left exhibited a weak signal, while the other small peaks gave no EPR signal (data not shown). Comparison of the EPR spectrum of the crude mixture (50 μM as estimated from A316, in magenta in the inset on the right) with a 100 μM 4-amino TEMPO standard (in green) revealed 65±10% spin content.

Supplementary Fig. 4 MALDI-ToF mass spectrum of TEMPO-HOCbl.

(a) MALDI-ToF mass spectrum of the major peak from the RP-HPLC of the crude product (as highlighted in Supplementary Fig. 3). The structure of TEMPO-HOCbl is shown as the inset. (b) Zoom in view of the MALDI-ToF mass spectrum. Prominent peaks in the spectra are close to the molecular mass of TEMPO-HOCbl (1543.61 g/mol).

Supplementary Fig. 5 LC-ESI-MS of TEMPO-HOCbl.

LC-ESI-MS data for the major peak from the RP-HPLC of the crude product (as highlighted in Supplementary Fig. 3). The peaks between m/z 1500-1600 are close to the molecular mass of TEMPO-HOCbl (1543.61 g/mol) and the peak at m/z 763.55 is close to the half of the molecular mass.

Supplementary Fig. 6 MTSL reduction in the E. coli cell suspension.

Cells (OD600 = ~80) were incubated with 500 μM MTSL (indicated by the dashed line) in the spin labeling buffer. Samples were periodically collected at the indicated time points, pelleted, and the MTSL concentration in the supernatant was monitored using RT CW EPR spectroscopy. For the zero-time sample, cells were pelleted immediately after mixing with MTSL (overall, which took an additional 6-7 min including centrifugation and EPR measurement). Error bars indicate the 15% error, which is typical for spin quantification using RT CW EPR spectroscopy. Similar trends were observed in independent experiments.

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Joseph, B., Jaumann, E.A., Sikora, A. et al. In situ observation of conformational dynamics and protein ligand–substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy. Nat Protoc 14, 2344–2369 (2019). https://doi.org/10.1038/s41596-019-0182-2

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