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European Biophysics Journal

, Volume 41, Issue 5, pp 475–482 | Cite as

Comparative analysis of the orientation of transmembrane peptides using solid-state 2H- and 15N-NMR: mobility matters

  • Stephan L. Grage
  • Erik Strandberg
  • Parvesh Wadhwani
  • Santiago Esteban-Martín
  • Jesús Salgado
  • Anne S. Ulrich
Original Paper

Abstract

Many solid-state nuclear magnetic resonance (NMR) approaches for membrane proteins rely on orientation-dependent parameters, from which the alignment of peptide segments in the lipid bilayer can be calculated. Molecules embedded in liquid-crystalline membranes, such as monomeric helices, are highly mobile, leading to partial averaging of the measured NMR parameters. These dynamic effects need to be taken into account to avoid misinterpretation of NMR data. Here, we compare two common NMR approaches: 2H-NMR quadrupolar waves, and separated local field 15N–1H polarization inversion spin exchange at magic angle (PISEMA) spectra, in order to identify their strengths and drawbacks for correctly determining the orientation and mobility of α-helical transmembrane peptides. We first analyzed the model peptide WLP23 in oriented dimyristoylphosphatidylcholine (DMPC) membranes and then contrasted it with published data on GWALP23 in dilauroylphosphatidylcholine (DLPC). We only obtained consistent tilt angles from the two methods when taking dynamics into account. Interestingly, the two related peptides differ fundamentally in their mobility. Although both helices adopt the same tilt in their respective bilayers (~20°), WLP23 undergoes extensive fluctuations in its azimuthal rotation angle, whereas GWALP23 is much less dynamic. Both alternative NMR methods are suitable for characterizing orientation and dynamics, yet they can be optimally used to address different aspects. PISEMA spectra immediately reveal the presence of large-amplitude rotational fluctuations, which are not directly seen by 2H-NMR. On the other hand, PISEMA was unable to define the azimuthal rotation angle in the case of the highly dynamic WLP23, though the helix tilt could still be determined, irrespective of any dynamics parameters.

Keywords

Membrane peptide orientation and dynamics WALP family peptides Geometric analysis of labeled alanines (GALA) 15N–1H PISEMA Models of peptide dynamics Selective isotope labeling 

Notes

Acknowledgments

This study was supported by the DFG Center for Functional Nanostructures CFN (E1.2) and the Spanish MICINN BFU2010-19118/BMC, financed in part by the European Regional Development Fund.

Supplementary material

249_2012_801_MOESM1_ESM.pdb (11 kb)
Structures representing the helix geometry used for the prediction of the solid-state NMR parameters are provided as pdb files in the Supplementary Materials. (PDB 10 kb)
249_2012_801_MOESM2_ESM.pdb (11 kb)
(PDB 10 kb)

References

  1. Afonin S, Grage SL, Ieronimo M, Parvesh W, Ulrich AS (2008) Temperature-dependent transmembrane insertion of the amphiphilic peptide PGLa in lipid bilayers, observed by solid state 19F NMR spectroscopy. J Am Chem Soc 130:16512–16514PubMedCrossRefGoogle Scholar
  2. Cornell BA, Separovic F, Baldassi AJ, Smith R (1988) Conformation and orientation of gramicidin A in oriented phospholipid bilayers measured by solid state carbon-13 NMR. Biophys J 53:67–76PubMedCrossRefGoogle Scholar
  3. Esteban-Martín S, Salgado J (2007) The dynamic orientation of membrane-bound peptides: bridging simulations and experiments. Biophys J 93:4278–4288PubMedCrossRefGoogle Scholar
  4. Esteban-Martín S, Strandberg E, Fuertes G, Ulrich AS, Salgado J (2009a) Influence of whole-body dynamics on 15N PISEMA NMR spectra of membrane peptides: a theoretical analysis. Biophys J 96:3233–3241PubMedCrossRefGoogle Scholar
  5. Esteban-Martín S, Giménez D, Fuertes G, Salgado J (2009b) Orientational landscapes of peptides in membranes: prediction of 2H NMR couplings in a dynamic context. Biochemistry 48:11441–11448PubMedCrossRefGoogle Scholar
  6. Esteban-Martín S, Strandberg E, Salgado J, Ulrich AS (2010) Solid state NMR analysis of peptides in membranes: influence of dynamics and labeling scheme. Biochim Biophys Acta 1798:252–257PubMedCrossRefGoogle Scholar
  7. Grage SL, Afonin S, Ulrich AS (2010) Dynamic transitions of membrane active peptides. Methods Mol Biol 618:183–207PubMedCrossRefGoogle Scholar
  8. Holt A, Koehorst RBM, Rutters-Meijneke T, Gelb MH, Rijkers DTS, Hemminga MA, Killian JA (2009) Tilt and rotation angles of a transmembrane model peptide as studied by fluorescence spectroscopy. Biophys J 97:2258–2266PubMedCrossRefGoogle Scholar
  9. Holt A, Rougier L, Reat V, Jolibois F, Saurel O, Czaplicki J, Killian JA, Milon A (2010) Order parameters of a transmembrane helix in a fluid bilayer: case study of a WALP peptide. Biophys J 98:1864–1872PubMedCrossRefGoogle Scholar
  10. Im W, Brooks CL III (2005) Interfacial folding and membrane insertion of designed peptides studied by molecular dynamics simulations. Proc Natl Acad Sci USA 102:6771–6776PubMedCrossRefGoogle Scholar
  11. Jo S, Im W (2011) Transmembrane helix orientation and dynamics: insights from ensemble dynamics with solid-state NMR observables. Biophys J 100:2913–2921PubMedCrossRefGoogle Scholar
  12. Killian JA, Salemink I, de Planque MR, Lindblom G, Koeppe RE II, Greathouse DV (1996) Induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane α-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans. Biochemistry 35:1037–1104PubMedCrossRefGoogle Scholar
  13. Kim T, Jo S, Im W (2011) Solid-state NMR ensemble dynamics as a mediator between experiment and simulation. Biophys J 100:2922–2928PubMedCrossRefGoogle Scholar
  14. Lee DK, Wei Y, Ramamoorthy A (2001) A two dimensional magic-angle decoupling and magic-angle turning solid-state NMR method: an application to study chemical shift tensors from peptides that are nonselectively labeled with 15N isotope. J Phys Chem B 105:4752–4762CrossRefGoogle Scholar
  15. Marassi FM, Opella SJ (2000) A solid-state NMR index of membrane protein structure and topology. J Magn Reson 144:156–161PubMedCrossRefGoogle Scholar
  16. Monticelli L, Tieleman DP, Fuchs PFJ (2010) Interpretation of 2H-NMR experiments on the orientation of the transmembrane helix WALP23 by computer simulations. Biophys J 99:1455–1464PubMedCrossRefGoogle Scholar
  17. Nevzorov AA, Opella SJ (2007) Selective averaging for high-resolution solid-state NMR spectroscopy of aligned samples. J Magn Reson 185:59–70PubMedCrossRefGoogle Scholar
  18. Özdirekcan S, Rijkers DTS, Liskamp RM, Killian JA (2005) Influence of flanking residues on tilt and rotation angles of transmembrane peptides in lipid bilayers. A solid-state 2H NMR study. Biochemistry 44:1004–1012PubMedCrossRefGoogle Scholar
  19. Özdirekcan S, Etchebest C, Killian JA, Fuchs PF (2007) On the orientation of a designed transmembrane peptide: toward the right tilt angle? J Am Chem Soc 129:15174–15181PubMedCrossRefGoogle Scholar
  20. Separovic F, Pax R, Cornell B (1993) NMR order parameter analysis of a peptide plane aligned in a lyotropic liquid crystal. Mol Phys 78:357–369CrossRefGoogle Scholar
  21. Smith R, Separovic F, Milne TJ, Whittaker A, Bennett FM, Cornell BA, Makriyannis A (1994) Structure and orientation of the pore-forming peptide melittin, in lipid bilayers. J Mol Biol 241:456–466PubMedCrossRefGoogle Scholar
  22. Strandberg E, Özdirekcan S, Rijkers DTS, van der Wel PCA, Koeppe RE II, Liskamp RM, Killian JA (2004) Tilt angles of transmembrane model peptides in oriented and non-oriented lipid bilayers as determined by 2H solid state NMR. Biophys J 86:3709–3721PubMedCrossRefGoogle Scholar
  23. Strandberg E, Tremouilhac P, Wadhwani P, Ulrich AS (2009a) Synergistic transmembrane insertion of the heterodimeric PGLa/magainin 2 complex studied by solid-state NMR. Biochim Biophys Acta 1788:1667–1679PubMedCrossRefGoogle Scholar
  24. Strandberg E, Esteban-Martín S, Salgado J, Ulrich AS (2009b) Orientation and dynamics of peptides in membranes calculated from 2H-NMR data. Biophys J 96:3223–3232PubMedCrossRefGoogle Scholar
  25. Strandberg E, Esteban-Martín S, Ulrich AS, Salgado J (2012) Hydrophobic mismatch of mobile transmembrane helices: Merging theory and experiments, Biochim Biophys Acta 1818:1242–1249Google Scholar
  26. van der Wel PCA, Strandberg E, Killian JA, Koeppe RE II (2002) Geometry and intrinsic tilt of a tryptophan-anchored transmembrane α-helix determined by 2H NMR. Biophys J 83:1479–1488PubMedCrossRefGoogle Scholar
  27. Vostrikov VV, Grant CV, Daily AE, Opella SJ, Koeppe RE II (2008) Comparison of “polarization inversion with spin exchange at magic angle” and “geometric analysis of labeled alanines” methods for transmembrane helix alignment. J Am Chem Soc 130:12584–12585PubMedCrossRefGoogle Scholar
  28. Vostrikov VV, Daily AE, Greathouse DV, Koeppe RE II (2010) Charged or aromatic anchor residue dependence of transmembrane peptide tilt. J Biol Chem 285:31723–31730PubMedCrossRefGoogle Scholar
  29. Walther TH, Grage SL, Roth N, Ulrich AS (2010) Membrane alignment of the pore-forming component TatAd of the twin-arginine translocase from Bacillus subtilis resolved by solid-state NMR spectroscopy. J Am Chem Soc 132:15945–15956PubMedCrossRefGoogle Scholar
  30. Wang J, Denny J, Tian C, Kim S, Mo Y, Kovacs F, Song Z, Nishimura K, Gan Z, Fu R, Quine JR, Cross TA (2000) Imaging membrane protein helical wheels. J Magn Reson 144:162–167PubMedCrossRefGoogle Scholar
  31. Wu CH, Ramamoorthy A, Opella SJ (1994) High-resolution heteronuclear dipolar solid-state NMR-spectroscopy. J Magn Reson A 109:270–272CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2012

Authors and Affiliations

  • Stephan L. Grage
    • 1
  • Erik Strandberg
    • 1
  • Parvesh Wadhwani
    • 1
  • Santiago Esteban-Martín
    • 2
  • Jesús Salgado
    • 3
  • Anne S. Ulrich
    • 1
  1. 1.Institute for Biological Interfaces (IBG-2) and Institute of Organic Chemistry and CFNKarlsruhe Institute of TechnologyKarlsruheGermany
  2. 2.Joint BSC-IRB Research Programme in Computational BiologyInstitute for Research in Biomedicine (IRB Barcelona), Parc Científic de BarcelonaBarcelonaSpain
  3. 3.Institute of Molecular ScienceUniversity of ValenciaPaterna (Valencia)Spain

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