Journal of Biomolecular NMR

, Volume 36, Issue 1, pp 55–71 | Cite as

Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase

  • Heather L. Frericks
  • Donghua H. Zhou
  • Lai Lai Yap
  • Robert B. Gennis
  • Chad M. Rienstra
Article

Abstract

Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo3 oxidase. The optimized protocol for expression and purification yields ∼5 mg of the enzymatically active, uniformly 13C,15N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The 13C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo3 oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly 13C,15N-labeled membrane protein.

Keywords

chemical shift correlation spectroscopy membrane protein recoupling sample preparation secondary structure 

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Notes

Acknowledgments

The funding for this work was provided by the University of Illinois (startup funds to C.M.R.), the NIH & NIGMS Roadmap Initiative (GM075937-01), and an Ullyot Fellowship to H.F. The authors would like to thank Dr. Paul Molitor (VOICE NMR Facility of School of Chemical Science, University of Illinois) for technical support and Drs. John Stringer and Charles Mullen (Varian, Inc.) for assistance with installation of the 750 MHz scroll resonator probe.

References

  1. Abramson J., Riistama S., Larsson G., Jasaitis A., Svensson-Ek M., Laakkonen L., Puustinen A., Iwata S., Wikstrom M., (2000). Nat. Struct. Biol. 7:910–917CrossRefGoogle Scholar
  2. Anderson C.A.F., Palmer A.G., Brunak S., Rost B., (2002). Structure 10:175–184CrossRefGoogle Scholar
  3. Baldus M., Petkova A.T., Herzfeld J.H., Griffin R.G., (1998). Mol. Phys. 95:1197–1207CrossRefADSGoogle Scholar
  4. Bennett A.E., Rienstra C.M., Auger M., Lakshmi K.V., Griffin R.G., (1995). J. Chem. Phys. 103:6951–6958CrossRefADSGoogle Scholar
  5. Caffrey M., (2003). J. Struct. Biol. 142:108–132CrossRefGoogle Scholar
  6. Chen P.S., Toribara T.Y., Warner H. (1956). Anal. Chem. 28:1756–1758CrossRefGoogle Scholar
  7. Cole H.B.R., Sparks S.W., Torchia D.A., (1988). Proc. Natl. Acad. Sci. 85:6362–6365CrossRefADSGoogle Scholar
  8. Das T.K., Tomson F.L., Gennis R.B., Gordon M., Rousseau D.L. (2001). Biophys. J. 80:2039–2045Google Scholar
  9. Distler A.M., Allison J., Hiser C., Qin L., Hilmi Y., Ferguson-Miller S. (2004). Eur. J. Mass Spectrom. 10:295–308CrossRefGoogle Scholar
  10. Egorova-Zachernyuk T.A., Hollander J., Fraser N., Gast P., Hoff A.J., Cogdell R., de Groot H.J.M., Baldus M. (2001). J. Biomol. NMR 19:243–253CrossRefGoogle Scholar
  11. Fahem S., Bowie J.U. (2002). J. Mol. Biol. 316:1–6CrossRefGoogle Scholar
  12. Franks W.T., Zhou D.H., Wylie B.J., Money B.G., Graesser D.T., Frericks H.L., Sahota G., Rienstra C.M. (2005). J. Am. Chem. Soc. 127:12291–12305CrossRefGoogle Scholar
  13. Garciahorsman J.A., Barquera B., Rumbley J., Ma J.X., Gennis R.B. (1994). J. Bacteriol. 176:5587–5600Google Scholar
  14. Goddard, T.D. and Kneller, D.G. (2004) Sparky 3.110 University of California, San FranciscoGoogle Scholar
  15. Harbison G.S., Herzfeld J., Griffin R.G. (1983). Biochemistry 22:1–5CrossRefGoogle Scholar
  16. Hatcher M.E., Hu J.G.G., Belenky M., Verdegem P., Lugtenburg J., Griffin R.G., Herzfeld J. (2002). Biophys. J. 82:1017–1029CrossRefGoogle Scholar
  17. Hellwig P., Yano T., Ohnishi T., Gennis R.B. (2002). Biochemistry 41:10675–10679CrossRefGoogle Scholar
  18. Hiller M., Krabben L., Vinothkumar K.R., Castellani F., van Rossum B.J., Kuhlbrandt W., Oschkinat H. (2005). Chem. Bio. Chem. 6:1679–1684Google Scholar
  19. Hohwy M., Rienstra C.M., Jaroniec C.P., Griffin R.G. (1999). J. Chem. Phys. 110:7983–7992CrossRefADSGoogle Scholar
  20. Hong M. (1999). J. Biomol. NMR 15:1–14CrossRefGoogle Scholar
  21. Hong M., Jakes K. (1999). J. Biomol. NMR 14:71–74CrossRefGoogle Scholar
  22. Hu J.G., Sun B.Q., Griffin R.G., Herzfeld J. (1995). Biophys. J. 68:A332Google Scholar
  23. Igumenova T.I., McDermott A.E., Zilm K.W., Martin R.W., Paulson E.K., Wand A.J. (2004a). J. Am. Chem. Soc. 126:6720–6727CrossRefGoogle Scholar
  24. Igumenova T.I., Wand A.J., McDermott A.E. (2004b). J. Am. Chem. Soc. 126:5323–5331CrossRefGoogle Scholar
  25. Jaroniec C.P., Lansing J.C., Tounge B.A., Belenky M., Herzfeld J., Griffin R.G. (2001). J. Am. Chem. Soc. 123:12929–12930CrossRefGoogle Scholar
  26. Krabben L., van Rossum B.J., Castellani F., Bocharov E., Schulga A.A., Arseniev A.S., Weise C., Hucho F., Oschkinat H. (2004). FEBS Lett. 564:319–324CrossRefGoogle Scholar
  27. Lemaster D.M., Cronan J.E. (1982). J. Biol. Chem. 257:1224–1230Google Scholar
  28. Li C., Mo Y., Hu J., Chekmenev E.Y., Tian C., Gao F.P., Fu R., Gor’kov P.L., Brey W.W., Cross T.A. (2006a). J. Mag. Res. 180:51–57ADSGoogle Scholar
  29. Li Y., Wylie B.J., Rienstra C.M. (2006b). J. Mag. Res. 179:206–216CrossRefADSGoogle Scholar
  30. Lorch M., Fahem S., Kaiser C., Weber I., Mason A.J., Bowie J.U., Glaubitz C. (2005). Chem. Bio. Chem. 6:1693–1700Google Scholar
  31. Luo W.B., Yao X.L., Hong M. (2005). J. Am. Chem. Soc., 127:6402–6408CrossRefGoogle Scholar
  32. Ma J.X., Puustinen A., Wikstrom M., Gennis R.B. (1998). Biochemistry 37:11806–11811CrossRefGoogle Scholar
  33. Martin R.W., Zilm K.W. (2003). J. Magn. Reson. 165:162–174CrossRefADSGoogle Scholar
  34. Marulanda D., Tasayco M.L., Cataldi M., Arriaran V., Polenova T. (2005). J. Phys. Chem. B 109:18135–18145CrossRefGoogle Scholar
  35. Meier B.H. (1992). Chem. Phys. Lett. 188:201–207CrossRefADSGoogle Scholar
  36. Miroux B., Walker J.E. (1996). J. Mol. Biol. 260:289–298CrossRefGoogle Scholar
  37. Morcombe C.R., Gaponenko V., Byrd R.A., Zilm K.W. (2005). J. Am. Chem. Soc. 127:397–404CrossRefGoogle Scholar
  38. Oldfield E. (2002). Ann. Rev. Phys. Chem. 53:349–378CrossRefGoogle Scholar
  39. Pauli J., Baldus M., van Rossum B., de Groot H., Oschkinat H. (2001). Chem. Bio. Chem. 2:272–281Google Scholar
  40. Pautsch A., Vogt J., Model K., Siebold C., Schulz G.E. (1999). Proteins 34:167–172CrossRefGoogle Scholar
  41. Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. (2004). J. Comput. Chem. 25:1605–1612CrossRefGoogle Scholar
  42. Puustinen A., Morgan J.E., Verkhovsky M., Thomas J.W., Gennis R.B., Wikstrom M. (1992). Biochemistry 31:10363–10369CrossRefGoogle Scholar
  43. Rumbley J.N., Nickels E.F., Gennis R.B. (1997). Biochim. Biophys. Acta 1340:131–142Google Scholar
  44. Schaefer J., Stejskal E.O. (1979). J. Magn. Reson. 34:443–447Google Scholar
  45. Seavey B.R., Farr E.A., Westler W.M., Markley J.L. (1991). J. Biomol. NMR 1:217–236CrossRefGoogle Scholar
  46. Sorgen P.L., Cahill S.M., Krueger-Koplin R.D., Krueger-Koplin S.T., Schenck C.C., Girvin M.E. (2002). Biochemistry 41:31–41CrossRefGoogle Scholar
  47. Stringer J.A., Bronnimann C.E., Mullen C.G., Zhou D.H., Stellfox S.A., Li Y., Williams E.H., Rienstra C.M. (2005). J. Magn. Reson. 173:40–48CrossRefADSGoogle Scholar
  48. Takegoshi K., Mizokami J., Terao T. (2001). Chem. Phys. Lett. 341:540–544CrossRefADSGoogle Scholar
  49. Thomas J.W., Puustinen A., Alben J.O., Gennis R.B., Wikstrom M. (1993). Biochemistry 32:10923–10928CrossRefGoogle Scholar
  50. Uchida T., Mogi T., Nakamura H., Kitagawa T. (2004). J. Biol. Chem. 279:53613–53620CrossRefGoogle Scholar
  51. van Gammeren A.J., Hulsbergen F.B., Hollander J.G., de Groot H.J.M. (2005). J. Biomol. NMR 31:279–293CrossRefGoogle Scholar
  52. Vanliemt W.B.S., Boender G.J., Gast P., Hoff A.J., Lugtenburg J., Degroot H.J.M. (1995). Biochemistry 34:10229–10236CrossRefGoogle Scholar
  53. Vinogradova O., Sonnichsen F., Sanders C.R. (1998). J. Biomol. NMR 11:381–386CrossRefGoogle Scholar
  54. Wishart D.S., Sykes B.D. (1994). J. Biomol. NMR 4:171–180CrossRefGoogle Scholar
  55. Zaslavsky D., Gennis R.B. (2000). Biochim. Biophys. Acta 1458:164–179CrossRefGoogle Scholar
  56. Zhang J., Osborne J.P., Gennis R.B., Wang X.T. (2004). Arch. Biochem. Biophys. 421:186–191CrossRefGoogle Scholar
  57. Zhou D.H., Kloepper K.D., Winter K.A., Rienstra C.M. (2006). J. Biomol. NMR 34:245–257CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Heather L. Frericks
    • 1
  • Donghua H. Zhou
    • 1
  • Lai Lai Yap
    • 2
  • Robert B. Gennis
    • 1
    • 2
    • 3
  • Chad M. Rienstra
    • 1
    • 2
    • 3
  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.Center for Biophysics and Computational BiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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