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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abramson J., Riistama S., Larsson G., Jasaitis A., Svensson-Ek M., Laakkonen L., Puustinen A., Iwata S., Wikstrom M., (2000). Nat. Struct. Biol. 7:910–917
Anderson C.A.F., Palmer A.G., Brunak S., Rost B., (2002). Structure 10:175–184
Baldus M., Petkova A.T., Herzfeld J.H., Griffin R.G., (1998). Mol. Phys. 95:1197–1207
Bennett A.E., Rienstra C.M., Auger M., Lakshmi K.V., Griffin R.G., (1995). J. Chem. Phys. 103:6951–6958
Caffrey M., (2003). J. Struct. Biol. 142:108–132
Chen P.S., Toribara T.Y., Warner H. (1956). Anal. Chem. 28:1756–1758
Cole H.B.R., Sparks S.W., Torchia D.A., (1988). Proc. Natl. Acad. Sci. 85:6362–6365
Das T.K., Tomson F.L., Gennis R.B., Gordon M., Rousseau D.L. (2001). Biophys. J. 80:2039–2045
Distler A.M., Allison J., Hiser C., Qin L., Hilmi Y., Ferguson-Miller S. (2004). Eur. J. Mass Spectrom. 10:295–308
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 19:243–253
Fahem S., Bowie J.U. (2002). J. Mol. Biol. 316:1–6
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. 127:12291–12305
Garciahorsman J.A., Barquera B., Rumbley J., Ma J.X., Gennis R.B. (1994). J. Bacteriol. 176:5587–5600
Goddard, T.D. and Kneller, D.G. (2004) Sparky 3.110 University of California, San Francisco
Harbison G.S., Herzfeld J., Griffin R.G. (1983). Biochemistry 22:1–5
Hatcher M.E., Hu J.G.G., Belenky M., Verdegem P., Lugtenburg J., Griffin R.G., Herzfeld J. (2002). Biophys. J. 82:1017–1029
Hellwig P., Yano T., Ohnishi T., Gennis R.B. (2002). Biochemistry 41:10675–10679
Hiller M., Krabben L., Vinothkumar K.R., Castellani F., van Rossum B.J., Kuhlbrandt W., Oschkinat H. (2005). Chem. Bio. Chem. 6:1679–1684
Hohwy M., Rienstra C.M., Jaroniec C.P., Griffin R.G. (1999). J. Chem. Phys. 110:7983–7992
Hong M. (1999). J. Biomol. NMR 15:1–14
Hong M., Jakes K. (1999). J. Biomol. NMR 14:71–74
Hu J.G., Sun B.Q., Griffin R.G., Herzfeld J. (1995). Biophys. J. 68:A332
Igumenova T.I., McDermott A.E., Zilm K.W., Martin R.W., Paulson E.K., Wand A.J. (2004a). J. Am. Chem. Soc. 126:6720–6727
Igumenova T.I., Wand A.J., McDermott A.E. (2004b). J. Am. Chem. Soc. 126:5323–5331
Jaroniec C.P., Lansing J.C., Tounge B.A., Belenky M., Herzfeld J., Griffin R.G. (2001). J. Am. Chem. Soc. 123:12929–12930
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. 564:319–324
Lemaster D.M., Cronan J.E. (1982). J. Biol. Chem. 257:1224–1230
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. 180:51–57
Li Y., Wylie B.J., Rienstra C.M. (2006b). J. Mag. Res. 179:206–216
Lorch M., Fahem S., Kaiser C., Weber I., Mason A.J., Bowie J.U., Glaubitz C. (2005). Chem. Bio. Chem. 6:1693–1700
Luo W.B., Yao X.L., Hong M. (2005). J. Am. Chem. Soc., 127:6402–6408
Ma J.X., Puustinen A., Wikstrom M., Gennis R.B. (1998). Biochemistry 37:11806–11811
Martin R.W., Zilm K.W. (2003). J. Magn. Reson. 165:162–174
Marulanda D., Tasayco M.L., Cataldi M., Arriaran V., Polenova T. (2005). J. Phys. Chem. B 109:18135–18145
Meier B.H. (1992). Chem. Phys. Lett. 188:201–207
Miroux B., Walker J.E. (1996). J. Mol. Biol. 260:289–298
Morcombe C.R., Gaponenko V., Byrd R.A., Zilm K.W. (2005). J. Am. Chem. Soc. 127:397–404
Oldfield E. (2002). Ann. Rev. Phys. Chem. 53:349–378
Pauli J., Baldus M., van Rossum B., de Groot H., Oschkinat H. (2001). Chem. Bio. Chem. 2:272–281
Pautsch A., Vogt J., Model K., Siebold C., Schulz G.E. (1999). Proteins 34:167–172
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. 25:1605–1612
Puustinen A., Morgan J.E., Verkhovsky M., Thomas J.W., Gennis R.B., Wikstrom M. (1992). Biochemistry 31:10363–10369
Rumbley J.N., Nickels E.F., Gennis R.B. (1997). Biochim. Biophys. Acta 1340:131–142
Schaefer J., Stejskal E.O. (1979). J. Magn. Reson. 34:443–447
Seavey B.R., Farr E.A., Westler W.M., Markley J.L. (1991). J. Biomol. NMR 1:217–236
Sorgen P.L., Cahill S.M., Krueger-Koplin R.D., Krueger-Koplin S.T., Schenck C.C., Girvin M.E. (2002). Biochemistry 41:31–41
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. 173:40–48
Takegoshi K., Mizokami J., Terao T. (2001). Chem. Phys. Lett. 341:540–544
Thomas J.W., Puustinen A., Alben J.O., Gennis R.B., Wikstrom M. (1993). Biochemistry 32:10923–10928
Uchida T., Mogi T., Nakamura H., Kitagawa T. (2004). J. Biol. Chem. 279:53613–53620
van Gammeren A.J., Hulsbergen F.B., Hollander J.G., de Groot H.J.M. (2005). J. Biomol. NMR 31:279–293
Vanliemt W.B.S., Boender G.J., Gast P., Hoff A.J., Lugtenburg J., Degroot H.J.M. (1995). Biochemistry 34:10229–10236
Vinogradova O., Sonnichsen F., Sanders C.R. (1998). J. Biomol. NMR 11:381–386
Wishart D.S., Sykes B.D. (1994). J. Biomol. NMR 4:171–180
Zaslavsky D., Gennis R.B. (2000). Biochim. Biophys. Acta 1458:164–179
Zhang J., Osborne J.P., Gennis R.B., Wang X.T. (2004). Arch. Biochem. Biophys. 421:186–191
Zhou D.H., Kloepper K.D., Winter K.A., Rienstra C.M. (2006). J. Biomol. NMR 34:245–257
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Heather L. Frericks and Lai Lai Yap contributed equally to this work.
Rights and permissions
About this article
Cite this article
Frericks, H.L., Zhou, D.H., Yap, L.L. et al. Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase. J Biomol NMR 36, 55–71 (2006). https://doi.org/10.1007/s10858-006-9070-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10858-006-9070-5