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Application of High-Pressure Electron Paramagnetic Resonance (EPR) Spectroscopy in Protein Science

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Abstract

High hydrostatic pressure at a few thousand bars has been found to offer an opportunity to probe protein conformational states which possess lower hydrostatic volumes and are often not well populated under the ambient pressure. An important contribution of Prof. Wayne Hubbell is to initiate EPR studies on proteins under elevated pressure via establishing the theoretical basis of high-pressure EPR, so that parameters from EPR measurements can be correlated to key protein structural factors, such as activation volume and molar compressibility. Prof. Hubbell also developed a series of experimental devices for the measurement of continuous wave (CW) and double electron–electron resonance (DEER) EPR under elevated pressure. These efforts have directly resulted in a series of high-pressure EPR studies on globular and transmembrane proteins. Via this review in the current Special Issue, we aim to briefly cover the most recent findings in high-pressure EPR and its applications in protein science. We will focus on what each technique can do in protein studies, how to use the technique to measure needed information, and what each measured parameter tells us. We will also discuss the combination of high-pressure EPR with other techniques and the future perspectives of the field. The ultimate goal is to broaden the application of high-pressure EPR and its combination with other high-pressure biophysical techniques to improve protein science research.

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References

  1. J. McCoy, W.L. Hubbell, High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins. Proc. Natl. Acad. Sci. 108, 1331–1336 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. M.T. Lerch, Z. Yang, C. Altenbach, W.L. Hubbell, Chapter Two—high-pressure EPR and site-directed spin labeling for mapping molecular flexibility in proteins, in Methods in Enzymology, ed. by P.Z. Qin, K. Warncke (Academic Press, 2015), pp. 29–57

  3. H. Li, K. Akasaka, Conformational fluctuations of proteins revealed by variable pressure NMR. Biochim. Biophys. Acta Prot. Proteom. 1764, 331–345 (2006)

    Article  CAS  Google Scholar 

  4. K. Akasaka, Probing Conformational fluctuation of proteins by pressure perturbation. Chem. Rev. 106, 1814–1835 (2006)

    Article  CAS  PubMed  Google Scholar 

  5. M.T. Lerch, C.J. López, Z. Yang, M.J. Kreitman, J. Horwitz, W.L. Hubbell, Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure. Proc. Natl. Acad. Sci. 112, E2437–E2446 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. G. Bouvignies, P. Vallurupalli, D.F. Hansen, B.E. Correia, O. Lange, A. Bah, R.M. Vernon, F.W. Dahlquist, D. Baker, L.E. Kay, Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature 477, 111–114 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. A.E. Eriksson, W.A. Baase, X.J. Zhang, D.W. Heinz, M. Blaber, E.P. Baldwin, B.W. Matthews, Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science 255, 178–183 (1992)

    Article  CAS  PubMed  Google Scholar 

  8. F.A.A. Mulder, A. Mittermaier, B. Hon, F.W. Dahlquist, L.E. Kay, Studying excited states of proteins by NMR spectroscopy. Nat. Struct. Biol. 8, 932–935 (2001)

    Article  CAS  PubMed  Google Scholar 

  9. K. Kuwata, H. Li, H. Yamada, C.A. Batt, Y. Goto, K. Akasaka, High pressure NMR reveals a variety of fluctuating conformers in β-lactoglobulin. J. Mol. Biol. 305, 1073–1083 (2001)

    Article  CAS  PubMed  Google Scholar 

  10. R. Kitahara, S. Yokoyama, K. Akasaka, NMR snapshots of a fluctuating protein structure: ubiquitin at 30bar–3kbar. J. Mol. Biol. 347, 277–285 (2005)

    Article  CAS  PubMed  Google Scholar 

  11. R. Kitahara, S. Sareth, H. Yamada, E. Ohmae, K. Gekko, K. Akasaka, High pressure NMR reveals active-site hinge motion of folate-bound escherichia coli dihydrofolate reductase. Biochemistry 39, 12789–12795 (2000)

    Article  CAS  PubMed  Google Scholar 

  12. R. Kitahara, H. Yamada, K. Akasaka, P.E. Wright, High pressure NMR reveals that apomyoglobin is an equilibrium mixture from the native to the unfolded. J. Mol. Biol. 320, 311–319 (2002)

    Article  CAS  PubMed  Google Scholar 

  13. R. Winter, J. Jonas, High Pressure Molecular Science (SpringerLink 1999)

  14. R.W. Peterson, A.J. Wand, Self-contained high-pressure cell, apparatus, and procedure for the preparation of encapsulated proteins dissolved in low viscosity fluids for nuclear magnetic resonance spectroscopy. Rev. Sci. Instr. 76, 094101 (2005)

    Article  Google Scholar 

  15. Y. Fu, V. Kasinath, V.R. Moorman, N.V. Nucci, V.J. Hilser, A.J. Wand, Coupled motion in proteins revealed by pressure perturbation. J. Am. Chem. Soc. 134, 8543–8550 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. N.V. Nucci, B. Fuglestad, E.A. Athanasoula, A.J. Wand, Role of cavities and hydration in the pressure unfolding of T4 lysozyme. Proc. Natl. Acad. Sci. 111, 13846–13851 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. M.T. Lerch, J. Horwitz, J. McCoy, W.L. Hubbell, Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin. Proc. Natl. Acad. Sci. 110, E4714–E4722 (2013)

    Article  PubMed  PubMed Central  Google Scholar 

  18. M.T. Lerch, Z. Yang, E.K. Brooks, W.L. Hubbell, Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron-electron resonance. Proc. Natl. Acad. Sci. 111, E1201–E1210 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. D.E. Budil, S. Lee, S. Saxena, J.H. Freed, Nonlinear-least-squares analysis of slow-motion EPR spectra in one and two dimensions using a modified levenberg-marquardt algorithm. J. Magn. Reson. A 120, 155–189 (1996)

    Article  CAS  Google Scholar 

  20. S. Stoll, A. Schweiger, EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006)

    Article  CAS  PubMed  Google Scholar 

  21. A. Gross, L. Columbus, K. Hideg, C. Altenbach, W.L. Hubbell, Structure of the KcsA potassium channel from streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment. Biochemistry 38, 10324–10335 (1999)

    Article  CAS  PubMed  Google Scholar 

  22. L. Columbus, T. Kálai, J. Jekö, K. Hideg, W.L. Hubbell, Molecular motion of spin labeled side chains in α-helices: analysis by variation of side chain structure. Biochemistry 40, 3828–3846 (2001)

    Article  CAS  PubMed  Google Scholar 

  23. C.J. López, M.R. Fleissner, Z. Guo, A.K. Kusnetzow, W.L. Hubbell, Osmolyte perturbation reveals conformational equilibria in spin-labeled proteins. Prot. Sci. 18, 1637–1652 (2009)

    Article  Google Scholar 

  24. C.J. López, S. Oga, W.L. Hubbell, Mapping molecular flexibility of proteins with site-directed spin labeling: a case study of myoglobin. Biochemistry 51, 6568–6583 (2012)

    Article  PubMed  Google Scholar 

  25. A.P. Todd, J. Cong, F. Levinthal, C. Levinthal, W.L. Hubbell, Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation. Prot. Struct. Funct. Bioinform. 6, 294–305 (1989)

    Article  CAS  Google Scholar 

  26. M.R. Fleissner, M.D. Bridges, E.K. Brooks, D. Cascio, T. Kálai, K. Hideg, W.L. Hubbell, Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy. Proc. Natl. Acad. Sci. 108, 16241–16246 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. K.M. Schultz, C.S. Klug, High-pressure EPR spectroscopy studies of the E. Coli lipopolysaccharide transport proteins LptA and LptC. Appl. Magn. Reson. 48, 1341–1353 (2017)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. M.T. Lerch, R.A. Matt, M. Masureel, M. Elgeti, K.K. Kumar, D. Hilger, B. Foys, B.K. Kobilka, W.L. Hubbell, Viewing rare conformations of the β2 adrenergic receptor with pressure-resolved DEER spectroscopy. Proc. Natl. Acad. Sci. 117, 31824 (2020)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. D.R. Davydov, Z. Yang, N. Davydova, J.R. Halpert, W.L. Hubbell, Conformational mobility in cytochrome P450 3A4 explored by pressure-perturbation EPR spectroscopy. Biophys. J. 110, 1485–1498 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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).

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Author notes

  1. Austin MacRae and Zoe Armstrong have contributed equally to this work.

Authors and Affiliations

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

    Austin MacRae, Zoe Armstrong, Mary Lenertz, Qiaobin Li, Li Feng & Zhongyu Yang

  2. Dilworth Glyndon Felton High School, Dilworth, MN, 56547, USA

    Aiden Forge

  3. California State University San Marcos, San Marcos, CA, 92096, USA

    Max Wang

  4. Department of Chemistry and Biochemistry, University of Alabama, Tuscaloosa, AL, 35487, USA

    Wenfang Sun

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  1. Austin MacRae
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Contributions

AM, ZA, and ZY wrote the main manuscript text and prepared figures. ML and QL assisted in the literature search and figure preparation. AF and MWLF, and WS assisted in the literature search and writing. All authors reviewed the manuscript.

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Correspondence to Zhongyu Yang.

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MacRae, A., Armstrong, Z., Lenertz, M. et al. Application of High-Pressure Electron Paramagnetic Resonance (EPR) Spectroscopy in Protein Science. Appl Magn Reson 55, 45–59 (2024). https://doi.org/10.1007/s00723-023-01573-4

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  • DOI: https://doi.org/10.1007/s00723-023-01573-4

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