Skip to main content

Lipid-protein nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides

Abstract

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). 15N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and 31P-NMR spectroscopy. The 2D 1H-15N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased 1HN line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. 15N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (∼40 nsec at 45°C) is indicative of additional peptide motions within the Aam-I/LPN complex.

This is a preview of subscription content, access via your institution.

Abbreviations

Aam-I:

antibiotic antiamoebin I from Emericellopsis minima

apoA-1:

apolipoprotein A-1

CRINEPT:

cross-correlated relaxation-enhanced polarization

1D:

one-dimensional

2D:

two-dimensional

DDM:

β-dodecyl maltoside

DHPC:

dihexanoyl phosphatidylcholine

DLPC:

dilauroyl phosphatidylcholine

DLPG:

dilauroyl phosphatidylglycerol

DMPC:

dimyristoyl phosphatidylcholine

DOPC:

dioleoyl phosphatidylcholine

DOPE:

dioleoyl phosphatidylethanolamine

DOPG:

dioleoyl phosphatidylglycerol

HMQC:

heteronuclear multiple quantum correlation

HSQC:

heteronuclear single quantum correlation

INEPT:

insensitive nuclei enhanced by polarization transfer

KcsA:

K+-channel from Streptomyces lividans

LMPC:

lysomyristoyl phosphatidylcholine

LMPG:

lysomyristoyl phosphatidylglycerol

LPN:

lipid-protein nanodisc

MP:

membrane protein

MSP:

fragment 44-243 of human apolipoprotein A1 (membrane scaffold protein)

POPC:

palmitoyloleoyl phosphatidylcholine

R St :

hydrodynamic radius of a particle, Stokes radius

TM:

transmembrane

TROSY:

transverse relaxation-optimized spectroscopy

Δν:

half-peak width of NMR band

ηXY :

rate of cross-correlation between dipole-dipole and chemical shift anisotropy relaxation of the 15N nucleus

τR :

effective rotational correlation time

References

  1. 1.

    Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids, John Wiley and Sons, New York.

    Google Scholar 

  2. 2.

    Cavanagh, J., Fairbrother, W. J., Palmer III, A. G., Skelton, N. J., and Rance, M. (2006) Protein NMR Spectroscopy: Principles and Practice, 2nd Edn., Academic Press.

  3. 3.

    Tugarinov, V., Kanelis, V., and Kay, L. E. (2006) Nat. Protoc., 1, 749–754.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Pervushin, K., Riek, R., Wider, G., and Wuthrich, K. (1997) Proc. Natl. Acad. Sci. USA, 94, 12366–12371.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Riek, R., Fiaux, J., Bertlesen, E. B., Horwich, A. L., and Wuthrich, K. (2002) J. Am. Chem. Soc., 124, 12144–12153.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Tugarinov, V., Choy, W. Y., Orekhov, V. Y., and Kay, L. E. (2005) Proc. Natl. Acad. Sci. USA, 102, 622–627.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Horst, R., Bertelsen, E. B., Fiaux, J., Wider, G., Horwich, A. L., and Wuthrich, K. (2005) Proc. Natl. Acad. Sci. USA, 102, 12748–12753.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Sanders, C. R., and Sonnichsen, F. (2006) Magn. Reson. Chem., 44, S24–S40.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Page, R. C., Moore, J. D., Nguyen, H. B., Sharma, M., Chase, R., Gao, F. P., Mobley, C. K., Sanders, C. R., Ma, L., Sonnichsen, F. D., Lee, S., Howell, S. C., Opella, S. J., and Cross, T. A. (2006) J. Struct. Funct. Genom., 7, 51–64.

    Article  CAS  Google Scholar 

  10. 10.

    Krueger-Koplin, R. D., Sorgen, P. L., Krueger-Koplin, S. T., Rivera-Torres, I. O., Cahill, S. M., Hicks, D. B., Grinius, L., Krulwich, T. A., and Girvin, M. E. (2004) J. Biomol. NMR, 28, 43–57.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Chou, J. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., and Bax, A. (2002) J. Am. Chem. Soc., 124, 2450–2451.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Li, L., Chen, J., Mishra, V. K., Kurtz, J. A., Cao, D., Klon, A. E., Harvey, S. C., Anantharamaiah, G. M., and Segrest, J. P. (2004) J. Mol. Biol., 343, 1293–1311.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Shih, A. Y., Denisov, I. G., Phillips, J. C., Sligar, S. G., and Schulten, K. (2005) Biophys. J., 88, 548–556.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol., 14, 861–868.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Matz, C. E., and Jonas, A. (1982) J. Biol. Chem., 257, 4535–4540.

    PubMed  CAS  Google Scholar 

  16. 16.

    Nath, A., Atkins, W. M., and Sligar, S. G. (2007) Biochemistry, 46, 2059–2069.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Bayburt, T. H., and Sligar, S. G. (2003) Prot. Sci., 12, 2476–2481.

    Article  CAS  Google Scholar 

  18. 18.

    Whorton, M. R., Bokoch, M. P., Rasmussen, S. G. F., Huang, B., Zare, R. N., Kobilka, B., and Sunahara, R. K. (2007) Proc. Natl. Acad. Sci. USA, 104, 7682–7687.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Banerjee, S., Huber, T., and Sakmar, T. P. (2008) J. Mol. Biol., 377, 1067–1081.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Denisov, I. G., McLean, M. A., Shaw, A. W., Grinkova, Y. V., and Sligar, S. G. (2005) J. Phys. Chem. B., 109, 15580–15588.

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Bayburt, T. H., Grinkova, Y. L., and Sligar, S. G. (2002) Nano Lett., 2, 853–856.

    Article  CAS  Google Scholar 

  22. 22.

    Denisov, I. G., Grinkova, Y. V., Lazarides, A. A., and Sligar, S. G. (2004) J. Am. Chem. Soc., 126, 3477–3487.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Lyukmanova, E. N., Shenkarev, Z. O., Paramonov, A. S., Sobol, A. G., Ovchinnikova, T. V., Chupin, V. V., Kirpichnikov, M. P., Blommers, M. J., and Arseniev, A. S. (2008) J. Am. Chem. Soc., 130, 2140–2141.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Nekrasova, O. V., Ignatova, A. A., Nazarova, A. I., Feofanov, A. V., Korolkova, Y. V., Boldyreva, E. F., Tagvei, A. I., Grishin, E. V., Arseniev, A. S., and Kirpichnikov, M. P. (2009) J. Neuroimmune Pharmacol., 4, 83–91.

    PubMed  Article  Google Scholar 

  25. 25.

    Heginbotham, L., Odessey, E., and Miller, C. (1997) Biochemistry, 36, 10335–10340.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Shenkarev, Z. O., Paramonov, A. S., Nadezhdin, K. D., Bocharov, E. V., Kudelina, I. A., Skladnev, D. A., Tagaev, A. A., Yakimenko, Z. A., Ovchinnikova, T. V., and Arseniev, A. S. (2007) Chem. Biodivers., 4, 1219–1242.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., and Sykes, B. D. (1995) J. Biomol. NMR, 6, 135–140.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Pearce, J. M., and Komoroski, R. A. (1993) Magn. Reson. Med., 29, 724–731.

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Lee, D., Hilty, C., Wider, G., and Wuthrich, K. (2006) J. Magn. Reson., 178, 72–76.

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Ortega, A., and de la Torre, J. G. (2003) J. Chem. Phys., 119, 9914–9919.

    Article  CAS  Google Scholar 

  31. 31.

    Carbone, M. A., and Macdonald, P. M. (1996) Biochemistry, 35, 3368–3378.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science, 280, 69–77.

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Chill, J. H., Louis, J. M., Miller, C., and Bax, A. (2006) Prot. Sci., 15, 684–698.

    Article  CAS  Google Scholar 

  34. 34.

    Baker, K. A., Tzitzilonis, C., Kwiatkowski, W., Choe, S., and Riek, R. (2007) Nat. Struct. Mol. Biol., 14, 1089–1095.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Chupin, V. V., Nekrasova, O. V., Kirpichnikov, M. P., and Arseniev, A. S. (2007) Patent of RF No. 2306319, September 20, 2007.

  36. 36.

    Schleucher, J., Schwendinger, M., Sattler, M., Schmidt, P., Schedletzky, O., Glaser, S. J., Sorensen, O. W., and Griesinger, C. (1994) J. Biomol. NMR, 4, 301–306.

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Nietlispach, D. (2005) J. Biomol. NMR, 31, 161–166.

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Palmer III, A. G., Kroenke, C. D., and Loria, J. P. (2001) Meth. Enzymol., 339, 204–238.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Z. O. Shenkarev.

Additional information

Original Russian Text © Z. O. Shenkarev, E. N. Lyukmanova, O. I. Solozhenkin, I. E. Gagnidze, O. V. Nekrasova, V. V. Chupin, A. A. Tagaev, Z. A. Yakimenko, T. V. Ovchinnikova, M. P. Kirpichnikov, A. S. Arseniev, 2009, published in Biokhimiya, 2009, Vol. 74, No. 7, pp. 933–945.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM09-026, May 10, 2009.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Shenkarev, Z.O., Lyukmanova, E.N., Solozhenkin, O.I. et al. Lipid-protein nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides. Biochemistry Moscow 74, 756–765 (2009). https://doi.org/10.1134/S0006297909070086

Download citation

Key words

  • nanodisc
  • apolipoprotein
  • high-density lipoprotein particle
  • membrane protein
  • membrane-active peptide
  • model membranes
  • NMR spectroscopy