Skip to main content
SpringerLink
Log in

Recent Applications of Triarylmethyl (TAM) Derivatives as Electron Paramagnetic Resonance (EPR) Spin Labels in Biomacromolecular Structural Studies

  • Review
  • Published:
Applied Magnetic Resonance Aims and scope Submit manuscript

Abstract

Triarylmethyl (TAM) radicals, also known as trityl radicals, have found wide applications in electron paramagnetic resonance (EPR), EPR imaging (EPRI), and dynamic nuclear polarization (DNP). Of particular interest is the use of TAM as a spin label/tag to study biomacromolecular structure and dynamics using various pulsed EPR techniques, a field initiated by the Hubbell and Schiemann groups. The long relaxation times of TAM spin labels as compared to the traditional nitroxide and paramagnetic metal ion spin labels/probes allow for inter-spin distance measurements under conditions above the cryogenic temperatures, leading to protein structure and conformational dynamics information under the physiological conditions. In addition, the high spectral intensity of TAM spin labels can enhance the percentage of spins being affected by microwave pulses commonly used in pulsed EPR spectroscopy, allowing for distance measurement in samples at low concentrations. The distinct spectral feature and minimal overlap with other commonly used spin centers also opens an avenue for orthogonal spin labeling and selective measurement of distances in protein samples. More recently, the enhanced protection of the radical center enhances the stability of the spin center under the reducing conditions in cells, leading to in-cell distance measurements, one further step toward distance measurement in proteins under the native conditions. The current review in this Special Issue aims to concisely highlight one of Prof. Wayne Hubbell’s contributions to spin labeling and EPR methodological development, followed by the applications of TAM spin labels in recent biomacromolecular studies. The goal is to generate further excitement in the use of TAM spin labels to probe biomacromolecular structure and dynamics for various purposes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Copyright 2012 American Chemical Society

Fig. 2
Fig. 3

Copyright 2015 American Chemical Society [26]

Fig. 4
Fig. 5

Similar content being viewed by others

Availability of Data and Materials

Not applicable.

References

  1. F. Hyodo, S. Matsumoto, N. Devasahayam, C. Dharmaraj, S. Subramanian, J.B. Mitchell, M.C. Krishna, Pulsed EPR imaging of nitroxides in mice. J. Magn. Reson. 197, 181–185 (2009)

    CAS  PubMed  Google Scholar 

  2. A.A. Bobko, I. Dhimitruka, J.L. Zweier, V.V. Khramtsov, Trityl radicals as persistent dual function ph and oxygen probes for in vivo electron paramagnetic resonance spectroscopy and imaging: concept and experiment. J. Am. Chem. Soc. 129, 7240–7241 (2007)

    CAS  PubMed  Google Scholar 

  3. I. Dhimitruka, O. Grigorieva, J.L. Zweier, V.V. Khramtsov, Synthesis, structure, and EPR characterization of deuterated derivatives of Finland trityl radical. Bioorg. Med. Chem. Lett. 20, 3946–3949 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. I. Dhimitruka, M. Velayutham, A.A. Bobko, V.V. Khramtsov, F.A. Villamena, C.M. Hadad, J.L. Zweier, Large-scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging. Bioorg. Med. Chem. Lett. 17, 6801–6805 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. T.J. Reddy, T. Iwama, H.J. Halpern, V.H. Rawal, General synthesis of persistent trityl radicals for EPR imaging of biological systems. J. Org. Chem. 67, 4635–4639 (2002)

    CAS  PubMed  Google Scholar 

  6. Z. Yang, Y. Liu, P. Borbat, J.L. Zweier, J.H. Freed, W.L. Hubbell, Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution. J. Am. Chem. Soc. 134, 9950–9952 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Google Scholar 

  8. W.L. Hubbell, C.J. López, C. Altenbach, Z. Yang, Technological advances in site-directed spin labeling of proteins. Curr. Opin. Struct. Biol. 23, 725–733 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. W.L. Hubbell, D.S. Cafiso, C. Altenbach, Identifying conformational changes with site-directed spin labeling. Nat. Struct. Biol. 7, 735–739 (2000)

    CAS  PubMed  Google Scholar 

  10. O. Krumkacheva, E. Bagryanskaya, EPR-based distance measurements at ambient temperature. J. Magn. Reson. 280, 117–126 (2017)

    CAS  PubMed  Google Scholar 

  11. V.M. Tormyshev, E.G. Bagryanskaya, Trityl radicals: synthesis, properties, and applications. Russ. Chem. Bull. 70, 2278–2297 (2021)

    CAS  Google Scholar 

  12. E.R. Georgieva, A.S. Roy, V.M. Grigoryants, P.P. Borbat, K.A. Earle, C.P. Scholes, J.H. Freed, 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)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. T. Schmidt, J. Jeon, W.-M. Yau, C.D. Schwieters, R. Tycko, G.M. Clore, Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca2+-ligated calmodulin. Proc. Natl. Acad. Sci. 119, e2122308119 (2022)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. D.I. Freedberg, P. Selenko, Live cell NMR. Ann. Rev. Biophys. 43, 171–192 (2014)

  15. C.M. Davis, J. Deutsch, M. Gruebele, An in vitro mimic of in-cell solvation for protein folding studies. Prot. Sci. 29, 1046–1054 (2020)

    Google Scholar 

  16. A. Bonucci, O. Ouari, B. Guigliarelli, V. Belle, E. Mileo, In-cell EPR: progress towards structural studies inside cells. ChemBioChem 21, 451–460 (2020)

    CAS  PubMed  Google Scholar 

  17. A.E. Smith, L.Z. Zhou, A.H. Gorensek, M. Senske, G.J. Pielak, In-cell thermodynamics and a new role for protein surfaces. Proc. Natl. Acad. Sci. 113, 1725–1730 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Z. Hasanbasri, M. Poncelet, H. Hunter, B. Driesschaert, S. Saxena, A new 13C trityl-based spin label enables the use of DEER for distance measurements. J. Magn. Reson. 347, 107363 (2023)

    CAS  PubMed  Google Scholar 

  19. A.A. Kuzhelev, O.A. Krumkacheva, G.Y. Shevelev, M. Yulikov, M.V. Fedin, E.G. Bagryanskaya, Room-temperature distance measurements using RIDME and the orthogonal spin labels trityl/nitroxide. Phys. Chem. Chem. Phys. 20, 10224–10230 (2018)

    CAS  PubMed  Google Scholar 

  20. S. Razzaghi, E.K. Brooks, E. Bordignon, W.L. Hubbell, M. Yulikov, G. Jeschke, EPR relaxation-enhancement-based distance measurements on orthogonally spin-labeled T4-lysozyme. ChemBioChem 14, 1883–1890 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. L. Garbuio, E. Bordignon, E.K. Brooks, W.L. Hubbell, G. Jeschke, M. Yulikov, Orthogonal spin labeling and Gd(iii)-nitroxide distance measurements on bacteriophage T4-lysozyme. J. Phys. Chem. B 117, 3145–3153 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. S. Ketter, B. Joseph, Gd3+–trityl–nitroxide triple labeling and distance measurements in the heterooligomeric cobalamin transport complex in the native lipid bilayers. J. Am. Chem. Soc. 145, 960–966 (2023)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. N. Fleck, C.A. Heubach, T. Hett, F.R. Haege, P.P. Bawol, H. Baltruschat, O. Schiemann, SLIM: a short-linked, highly redox-stable trityl label for high-sensitivity in-cell EPR distance measurements. Angew. Chem. Int. Ed. 59, 9767–9772 (2020)

    CAS  Google Scholar 

  24. Y. Yang, B.-B. Pan, X. Tan, F. Yang, Y. Liu, X.-C. Su, D. Goldfarb, In-cell trityl–trityl distance measurements on proteins. J. Phys. Chem. Lett. 11, 1141–1147 (2020)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. J.J. Jassoy, A. Berndhäuser, F. Duthie, S.P. Kühn, G. Hagelueken, O. Schiemann, Versatile trityl spin labels for nanometer distance measurements on biomolecules in vitro and within cells. Angew. Chem. Int. Ed. 56, 177–181 (2017)

    CAS  Google Scholar 

  26. G.Y. Shevelev, O.A. Krumkacheva, A.A. Lomzov, A.A. Kuzhelev, D.V. Trukhin, O.Y. Rogozhnikova, V.M. Tormyshev, D.V. Pyshnyi, M.V. Fedin, E.G. Bagryanskaya, Triarylmethyl labels: toward improving the accuracy of EPR nanoscale distance measurements in DNAs. J. Phys. Chem. B 119, 13641–13648 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Google Scholar 

  28. G.Y. Shevelev, O.A. Krumkacheva, A.A. Lomzov, A.A. Kuzhelev, O.Y. Rogozhnikova, D.V. Trukhin, T.I. Troitskaya, V.M. Tormyshev, M.V. Fedin, D.V. Pyshnyi, E.G. Bagryanskaya, Physiological-temperature distance measurement in nucleic acid using triarylmethyl-based spin labels and pulsed dipolar EPR spectroscopy. J. Am. Chem. Soc. 136, 9874–9877 (2014)

    CAS  PubMed  Google Scholar 

  29. Z. Yang, G. Jiménez-Osés, C.J. López, M.D. Bridges, K.N. Houk, W.L. Hubbell, Long-range distance measurements in proteins at physiological temperatures using saturation recovery EPR spectroscopy. J. Am. Chem. Soc. 136, 15356–15365 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. S. Jun, J.S. Becker, M. Yonkunas, R. Coalson, S. Saxena, Unfolding of alanine-based peptides using electron spin resonance distance measurements. Biochemistry 45, 11666–11673 (2006)

    CAS  PubMed  Google Scholar 

  31. H.S. McHaourab, K.J. Oh, C.J. Fang, W.L. Hubbell, Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling. Biochemistry 36, 307–316 (1997)

  32. D.J. Hirsh, W.F. Beck, J.B. Innes, G.W. Brudvig, Using saturation-recovery EPR to measure distances in proteins: applications to photosystem II. Biochemistry 31, 532–541 (1992)

    CAS  PubMed  Google Scholar 

  33. S.S. Eaton, G.R. Eaton, in Saturation Recovery EPR, ed. by S.S. Eaton, L.J. Berliner, G.R. Eaton. Biological Magnetic Resonance, vol 24: Biomedical EPR, Part B: Methodology, Instrumentation, and Dynamics (Springer, New York City, 2005)

  34. A.V. Kulikov, G.I. Likhtenstein, The use of spin relaxation phenomena in the investigation of the structure of model and biological systems by the method of spin labels. Adv. Mol. Relax. Interact. Process. 10, 47–79 (1977)

    CAS  Google Scholar 

  35. A.A. Kuzhelev, D.V. Trukhin, O.A. Krumkacheva, R.K. Strizhakov, O.Y. Rogozhnikova, T.I. Troitskaya, M.V. Fedin, V.M. Tormyshev, E.G. Bagryanskaya, Room-temperature electron spin relaxation of triarylmethyl radicals at the X- and Q-bands. J. Phys. Chem. B 119, 13630–13640 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Z. Yang, M. Bridges, C. Lopez, O.Y. Rogozhnikova, D. Trukhin, E. Brooks, V. Tormyshev, H. Halpern, W. Hubbell, A triarylmethyl spin label for long-range distance measurement at physiological temperature using T1 relaxation enhancement. J. Magn. Reson. 269, 50–54 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. N.C. Kunjir, G.W. Reginsson, O. Schiemann, S.T. Sigurdsson, Measurements of short distances between trityl spin labels with CW EPR, DQC and PELDOR. Phys. Chem. Chem. Phys. 15, 19673–19685 (2013)

    CAS  PubMed  Google Scholar 

  38. M.R. Fleissner, E.M. Brustad, T. Kálai, C. Altenbach, D. Cascio, F.B. Peters, K. Hideg, S. Peuker, P.G. Schultz, W.L. Hubbell, Site-directed spin labeling of a genetically encoded unnatural amino acid. Proc. Natl. Acad. Sci. 106, 21637–21642 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. J. Voss, J. Wu, W.L. Hubbell, V. Jacques, C.F. Meares, H.R. Kaback, Helix packing in the lactose permease of Escherichia coli: distances between site-directed nitroxides and a lanthanide. Biochemistry 40, 3184–3188 (2001)

    CAS  PubMed  Google Scholar 

  40. B. Joseph, V.M. Tormyshev, O.Y. Rogozhnikova, D. Akhmetzyanov, E.G. Bagryanskaya, T.F. Prisner, Selective high-resolution detection of membrane protein–ligand interaction in native membranes using trityl–nitroxide PELDOR. Angew. Chem. Int. Ed. 55, 11538–11542 (2016)

    CAS  Google Scholar 

  41. A. Gamble Jarvi, X. Bogetti, K. Singewald, S. Ghosh, S. Saxena, Going the dHis-tance: site-directed Cu2+ labeling of proteins and nucleic acids. Acc. Chem. Res. 54, 1481–1491(2021)

  42. J. Casto, A. Mandato, S. Saxena, dHis-troying barriers: deuteration provides a pathway to increase sensitivity and accessible distances for Cu2+ labels. J. Phys. Chem. Lett. 12, 4681–4685 (2021)

    CAS  PubMed  Google Scholar 

  43. K. Singewald, X. Bogetti, K. Sinha, G.S. Rule, S. Saxena, Double histidine based EPR measurements at physiological temperatures permit site-specific elucidation of hidden dynamics in enzymes. Angew. Chem. Int. Ed. 59, 23040–23044 (2020)

    CAS  Google Scholar 

  44. M. Ji, S. Ruthstein, S. Saxena, Paramagnetic metal ions in pulsed ESR distance distribution measurements. Acc. Chem. Res. 47, 688–695 (2014)

    CAS  PubMed  Google Scholar 

  45. A. Giannoulis, Y. Yang, Y.-J. Gong, X. Tan, A. Feintuch, R. Carmieli, T. Bahrenberg, Y. Liu, X.-C. Su, D. Goldfarb, DEER distance measurements on trityl/trityl and Gd(iii)/trityl labelled proteins. Phys. Chem. Chem. Phys. 21, 10217–10227 (2019)

    CAS  PubMed  Google Scholar 

  46. A. Meyer, J.J. Jassoy, S. Spicher, A. Berndhäuser, O. Schiemann, Performance of PELDOR, RIDME, SIFTER, and DQC in measuring distances in trityl based bi- and triradicals: exchange coupling, pseudosecular coupling and multi-spin effects. Phys. Chem. Chem. Phys. 20, 13858–13869 (2018)

    CAS  PubMed  Google Scholar 

  47. A. Kuzhelev, D. Akhmetzyanov, V. Denysenkov, G. Shevelev, O. Krumkacheva, E. Bagryanskaya, T. Prisner, High-frequency pulsed electron–electron double resonance spectroscopy on DNA duplexes using trityl tags and shaped microwave pulses. Phys. Chem. Chem. Phys. 20, 26140–26144 (2018)

    CAS  PubMed  Google Scholar 

  48. D. Akhmetzyanov, P. Schöps, A. Marko, N.C. Kunjir, S.T. Sigurdsson, T.F. Prisner, Pulsed EPR dipolar spectroscopy at Q- and G-band on a trityl biradical. Phys. Chem. Chem. Phys. 17, 24446–24451 (2015)

    CAS  PubMed  Google Scholar 

  49. M. Bretschneider, P.E. Spindler, O.Y. Rogozhnikova, D.V. Trukhin, B. Endeward, A.A. Kuzhelev, E. Bagryanskaya, V.M. Tormyshev, T.F. Prisner, Multiquantum counting of trityl radicals. J. Phys. Chem. Lett. 11, 6286–6290 (2020)

    CAS  PubMed  Google Scholar 

  50. K.-N. Hu, V.S. Bajaj, M. Rosay, R.G. Griffin, High-frequency dynamic nuclear polarization using mixtures of TEMPO and trityl radicals. J. Chem. Phys. 126, 044512 (2007)

    PubMed  Google Scholar 

Download references

Acknowledgements

This work is supported by the National Science Foundation (NSF MCB 1942596 and NSF CBET 2217474 to Z. Y.).

Funding

This work is supported by the National Science Foundation (NSF MCB 1942596 and NSF CBET 2217474 to Z. Y.).

Author information

Author notes

  1. Zoe Armstrong and Austin MacRae contribute equally.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58102, USA

    Zoe Armstrong, Austin MacRae, Mary Lenertz, Qiaobin Li, Grace Blair, William Brown, Li Feng, Pinjing Zhao & Zhongyu Yang

Authors
  1. Zoe Armstrong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Austin MacRae
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mary Lenertz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qiaobin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Grace Blair
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. William Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Li Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pinjing Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhongyu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z. A., A. M., P. Z., and Z. Y. wrote the main manuscript text and prepared figures. M.L. and Q.L. assisted in literature search and figure preparation. G. B. and W. B. and L. F. assisted in literature search and writing. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Pinjing Zhao or Zhongyu Yang.

Ethics declarations

Conflict of Interest

The authors claim no competing interests.

Ethical Approval

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Armstrong, Z., MacRae, A., Lenertz, M. et al. Recent Applications of Triarylmethyl (TAM) Derivatives as Electron Paramagnetic Resonance (EPR) Spin Labels in Biomacromolecular Structural Studies. Appl Magn Reson 55, 29–44 (2024). https://doi.org/10.1007/s00723-023-01567-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00723-023-01567-2

Search

Navigation