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Measuring distances in proteins by saturation-recovery EPR

Nature Protocols volume 2, pages 1770–1781 (2007)Cite this article

Abstract

We describe a protocol for detecting electron spin–spin interactions between a radical and a metal ion in a protein or protein complex by saturation-recovery electron paramagnetic resonance (EPR). This protocol can be used with a protein containing an endogenous metal center and either an endogenous or synthetic radical species. We suggest a two-step approach whereby dipole–dipole or exchange interactions are first detected by continuous-wave EPR experiments and then quantified by saturation-recovery EPR. The latter measurements make it possible to measure long distances to within a few Ångstroms. The protocol for making distance measurements by saturation-recovery EPR will take approximately 6 days to complete.

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Figure 1: Energy diagram for radicals with a single unpaired electron (S = 1/2).
Figure 2: The interspin vector r forms an angle θ with the static magnetic field H0.
Figure 3: In the absence of electron spin–spin interactions, the saturation-recovery transients of tyrosine-based radicals are single-exponential.
Figure 4: Log–log plot showing microwave progressive power saturation data for EPR signals from (▪) the tyrosine radical YD• in PSII depleted of manganese, (□) the UV-generated L-tyrosine radical and (•) the tyrosine radical in RNR from Escherichia coli at 7.0 K.
Figure 5: The anatomy of a CW saturation-recovery experiment.
Figure 6: The relaxation behavior of the UV-generated tyrosine radical and the tyrosyl radical of the B2 subunit of RNR as a function of temperature.
Figure 7: Saturation-recovery transient from the radical P+ in the bacterial reaction center at 10 K.
Figure 8: Plotting of the dipolar rate constants k1d versus temperature for P+ (□), the cation radical of the special pair in photosynthetic reaction centers of Rb. sphaeroides, and for the tyrosine radical YD• (▪) in Mn-depleted PSII. Since the P+–Fe(II) distance, r, is shorter than the YD•–Fe(II) distance, the dipolar rate constant for P+ is greater at each temperature.

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References

  1. Lakshmi, K. & Brudvig, G. Pulsed electron paramagnetic resonance methods for macromolecular structure determination. Curr. Opin. Struct. Biol. 11, 523–531 (2003).

    Article  Google Scholar 

  2. Berliner L.J., Eaton G.R., & Eaton S.S. (eds.) Distance Measurements in Biological Systems by EPR (Kluwer Academic/Plenum Publishers, New York, 2000).

    Google Scholar 

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

    Article  CAS  PubMed Central  Google Scholar 

  4. Hirsh, D.J., Beck, W.F., Lynch, J.B., Que, L., Jr . & Brudvig, G.W. Using saturation-recovery EPR to measure exchange couplings in proteins: application to ribonucleotide reductase. J. Am. Chem. Soc. 114, 7475–7481 (1992).

    Article  CAS  Google Scholar 

  5. MacArthur, R. et al. Component B binding to the soluble methane monooxygenase hydroxylase by saturation-recovery EPR spectroscopy of spin-labeled MMOB. J. Am. Chem. Soc. 124, 13392–13393 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  6. Clegg, R.M. Fluorescence resonance energy transfer. in Fluorescence Imaging Spectroscopy and Microscopy Vol. 137 (eds. Wang, X.F. & Herman, B.) 179–251 (Wiley, New York, 1996).

    Google Scholar 

  7. Stryer, L. Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846 (1978).

    Article  CAS  PubMed Central  Google Scholar 

  8. Förster, T. Delocalized excitation and excitation transfer. in Modern Quantum Chemistry Vol. 3 (ed. Sinanoglu, O.) 93–137 (Academic Press Inc., New York, 1965).

    Google Scholar 

  9. Borbat, P.B. & Freed, J.H. Double-quantum ESR and distance measurements. in Distance Measurements in Biological Systems by EPR Vol. 19 (eds. Berliner, L.J., Eaton, G.R. & Eaton, S.S.) 383–456 (Kluwer Academic/Plenum Publishers, New York, 2000).

    Google Scholar 

  10. Raitsimring, A. “2 + 1” pulse sequence as applied for distance and spatial distribution measurements of paramagnetic centers. in Distance Measurements in Biological Systems by EPR Vol. 19 (eds. Berliner, L.J., Eaton, G.R. & Eaton, S.S.) 461–490 (Kluwer Academic/Plenum Publishers, New York, 2000).

    Google Scholar 

  11. Jeschke, G., Pannier, M. & Spiess, H.W. Distance measurements in biological systems by EPR. in Distance Measurements in Biological Systems by EPR Vol. 19 (eds. Berliner, L.J., Eaton, G.R. & Eaton, S.S.) 493–511 (Kluwer Academic/Plenum Publishers, New York, 2000).

    Google Scholar 

  12. Berliner L.J. (ed.) Spin Labeling 444 (Springer, New York, 2007).

    Google Scholar 

  13. Berliner L.J. (ed.) Spin Labeling II: Theory and Applications (Academic Press, New York, 1979).

    Google Scholar 

  14. Berliner L.J., (ed.) Spin Labelling 605 (Academic Press, New York, 1976).

    Google Scholar 

  15. Bloembergen, N. The interaction of nuclear spins in a crystalline lattice. Physica (The Hague) 15, 386–426 (1949).

    Article  CAS  Google Scholar 

  16. Bloembergen, N., Purcell, E.M. & Pound, R.V. Relaxation effects in nuclear magnetic resonance absorption. Phys. Rev. 73, 679–712 (1948).

    Article  CAS  Google Scholar 

  17. Bloembergen, N., Shapiro, S., Pershan, P.S. & Artman, J.O. Cross relaxation in spin systems. Phys. Rev. 114, 445–459 (1959).

    Article  CAS  Google Scholar 

  18. Abragam, A. Overhauser effect in nonmetals. Phys. Rev. 98, 1729–35 (1955).

    Article  CAS  Google Scholar 

  19. Kulikov, A.V. & Likhtenstein, G.I. Adv. Mol. Relax. Interact. Processes 10, 47–79 (1977).

    Article  CAS  Google Scholar 

  20. Goodman, G. & Leigh, J.S. Jr. Distance between the visible copper and cytochrome a in bovine heart cytochrome oxidase. Biochemistry 24, 2310–2317 (1985).

    Article  CAS  PubMed Central  Google Scholar 

  21. Rakowsky, M., More, K., Kulikov, A., Eaton, G. & Eaton, S. Time-domain electron paramagnetic resonance as a probe of electron–electron spin–spin interaction in spin-labeled low-spin iron porphyrins. J. Am. Chem. Soc. 117, 2049–2057 (1995).

    Article  CAS  Google Scholar 

  22. Eaton, G.R. & Eaton, S.S. Relaxation times of organic radicals and transition metal ions. in Distance Measurements in Biological Systems by EPR Vol. 19 (eds. Berliner, L.J., Eaton, G.R. & Eaton, S.S.) 29–154 (Kluwer Academic/Plenum Publishers, New York, 2000).

    Google Scholar 

  23. Debus, R.J., Feher, G. & Okamura, M.Y. Iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26.1: characterization and reconstitution with Fe2+, Mn2+, Co2+, Ni2+, Cu2+ and Zn2+. Biochemistry 25, 2276–2287 (1986).

    Article  CAS  PubMed Central  Google Scholar 

  24. Zhou, Y., Bowler, B., Lynch, K., Eaton, S. & Eaton, G. Interspin distances in spin-labeled metmyoglobin variants determined by saturation recovery EPR. Biophys. J. 79, 1039–1052 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  25. Seiter, M., Budker, V., Du, J.-L., Eaton, G.R. & Eaton, S.S. Interspin distances determined by time domain EPR of spin-labeled high-spin methemoglobin. Inorg. Chim. Acta 273, 354–366 (1998).

    Article  CAS  Google Scholar 

  26. Hirsh, D.J. & Brudvig, G.W. Long-range electron spin–spin interactions in the bacterial photosynthetic reaction center. J. Phys. Chem. 97, 13216–13222 (1993).

    Article  CAS  Google Scholar 

  27. Portis, A.M. Electronic structure of F centers: saturation of the electron spin resonance. Phys. Rev. 91, 1071–1078 (1953).

    Article  CAS  Google Scholar 

  28. Castner, T.G. Jr. Saturation of the paramagnetic resonance of a V center. Phys. Rev. 115, 1506–1515 (1959).

    Article  CAS  Google Scholar 

  29. Galli, C., Innes, J., Hirsh, D. & Brudvig, G. Effects of dipole–dipole interactions on microwave progressive power saturation of radicals in proteins. J. Magn. Reson. B 110, 284–287 (1996).

    Article  CAS  PubMed Central  Google Scholar 

  30. Voss, J., Hubbell, W.L. & Kaback, H.R. Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli. Proc. Natl. Acad. Sci. USA 92, 12300–12303 (1995).

    Article  CAS  PubMed Central  Google Scholar 

  31. Roberts, J.D. ABCs of FT-NMR (University Science Books, Sausalito, CA, 2000).

    Google Scholar 

  32. Harris, R.K. Nuclear Magnetic Resonance Spectroscopy: A Physicochemical View (Wiley, New York, 1986).

    Google Scholar 

  33. Weil, J.A., Bolton, J.R. & Wertz, J.E. Electron Paramagnetic Resonance: Elementary Theory and Practical Applications 568 (Wiley, New York, 1994).

    Google Scholar 

  34. Symons, M.M.C. Chemical and Biochemical Aspects of Electron-Spin Resonance Spectroscopy 190 (John Wiley, New York, 1978).

    Google Scholar 

  35. Hyde, J.S., Swartz, H.M. & Antholine, W.E. in Spin Labeling II: Theory and Applications (ed. Berliner, L.J.) 71–113 (Academic Press, New York, 1979).

    Book  Google Scholar 

  36. Hyde, J.S. Saturation recovery methodology. in Time-domain Electron Spin Resonance (eds. Kevan, L. & Schwartz, R.N.) 1–30 (Wiley, New York, 1979).

    Google Scholar 

  37. Yamauchi, T., Mino, H., Matsukawa, T., Kawamori, A. & Ono, T. Parallel polarization electron paramagnetic resonance studies of the S1-state manganese cluster in the photosynthetic oxygen-evolving system. Biochemistry 36, 7520–7526 (1997).

    Article  CAS  PubMed Central  Google Scholar 

  38. Bizzarri, A.R. & Cannistraro, S. Solvent modulation of the structural heterogeneity in FeIII myoglobin samples: a low temperature EPR investigation. Eur. Biophys. J. 22, 259–267 (1993).

    Article  CAS  PubMed Central  Google Scholar 

  39. Berthold, D.A., Babcock, G.T. & Yocum, C.F. A highly resolved, oxygen-evolving photosystem II preparation from spinach thylakoid membranes. FEBS Lett. 134, 231–234 (1981).

    Article  CAS  Google Scholar 

  40. Pikal-Cleland, K.A., Rodriguez-Hornedo, N., Amidon, G.L. & Carpenter, J.F. Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric beta-galactosidase. Arch. Biochem. Biophys. 384, 398–406 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  41. Eaton, S.S. & Eaton, G.R. Irradiated fused-quartz standard sample for time-domain EPR. J. Magn. Reson. A 102, 354–356 (1993).

    Article  CAS  Google Scholar 

  42. Sahlin, M. et al. Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase. Biochemistry 26, 5541–5548 (1987).

    Article  CAS  PubMed Central  Google Scholar 

  43. Allen, J.P., Feher, G., Yeates, T.O., Komiya, H. & Rees, D.C. Structure of the reaction center from Rhodobacter sphaeroides R-26: protein–cofactor (quinones and Fe2+) interactions. Proc. Natl. Acad. Sci. USA 85, 8487–8491 (1988).

    Article  CAS  PubMed Central  Google Scholar 

  44. Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J. & Iwata, S. Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831–1838 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  45. Loll, B., Kern, J., Saenger, W., Zouni, A. & Biesiadka, J. Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature (London, United Kingdom) 438, 1040–1044 (2005).

    Article  CAS  Google Scholar 

  46. Hirsh, D.J. Using Saturation-recovery EPR to Measure Distances and Exchange Couplings in Proteins PhD thesis, Yale University (1993).

    Google Scholar 

  47. Du, J.-L., Eaton, G.R. & Eaton, S.S. Temperature, orientation, and solvent dependence of electron spin–lattice relaxation rates for nitroxyl radicals in glassy solvents and doped solids. J. Magn. Reson. A 115, 213–221 (1995).

    Article  CAS  Google Scholar 

  48. Eaton, S.S. & Eaton, G.R. Determination of distances based on T1 and Tm effects. in Distance Measurements in Biological Systems by EPR Vol. 19 (eds. Berliner, L.J., Eaton, G.R. & Eaton, S.S.) 348–378 (Kluwer Academic/Plenum Publishers, New York, 2000).

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Chemistry, The College of New Jersey, PO Box 7718, Ewing, 08628, New Jersey, USA

    Donald J Hirsh

  2. Department of Chemistry, Yale University, 225 Prospect Street, PO Box 208107, New Haven, 06520, Connecticut, USA

    Gary W Brudvig

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  1. Donald J Hirsh
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Correspondence to Donald J Hirsh.

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Supplementary information

Supplementary Fig. 1

Spectral diffusion in the inhomogeneously broadened EPR signal (PDF 80 kb)

Supplementary Discussion

Why saturation-recovery (and not inversion recovery)? (PDF 26 kb)

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Hirsh, D., Brudvig, G. Measuring distances in proteins by saturation-recovery EPR. Nat Protoc 2, 1770–1781 (2007). https://doi.org/10.1038/nprot.2007.255

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