European Biophysics Journal

, Volume 36, Issue 4–5, pp 393–404

Solid-state NMR characterization of the putative membrane anchor of TWD1 from Arabidopsis thaliana

  • Holger A. Scheidt
  • Alexander Vogel
  • Andreas Eckhoff
  • Bernd W. Koenig
  • Daniel Huster


Structure and membrane interaction of a 31 amino acid residue fragment of the membrane bound FKBP-like protein twisted dwarf 1 (TWD1) from Arabidopsis thaliana was investigated by solid-state NMR spectroscopy. The studied peptide TWD1(335–365) contained the putative membrane anchor of the protein (residues 339–357) that was previously predicted by sequence hydrophobicity analysis. The TWD1 peptide was synthesized by standard solid phase peptide synthesis and contained three uniformly 13C- and 15N-labelled residues (Phe 340, Val 350, Ala 364). The peptide was incorporated into either multilamellar vesicles or oriented planar membranes composed of an equimolar ternary phospholipid mixture (POPC, POPE, POPG), where the POPC was sn-1 chain-deuterated. 31P NMR spectra of the membrane in the absence and in the presence of the peptide showed axially symmetric powder patterns indicative of a lamellar bilayer phase. Further, the addition of peptide caused a decrease in the lipid hydrocarbon chain order as indicated by reduced quadrupolar splittings in the 2H NMR spectra of the POPC in the membrane. The conformation of TWD1(335–365) was investigated by 13C cross-polarization magic-angle spinning NMR spectroscopy. At a temperature of −30°C all peptide signals were resolved and could be fully assigned in two-dimensional proton-driven 13C spin diffusion and 13C single quantum/double quantum correlation experiments. The isotropic chemical shift values for Phe 340 and Val 350 exhibited the signature of a regular α-helix. Chemical shifts typical for a random coil conformation were observed for Ala 364 located close to the C-terminus of the peptide. Static 15N NMR spectra of TWD1(335–365) in mechanically aligned lipid bilayers demonstrated that the helical segment of TWD1(335–365) adopts an orientation perpendicular to the membrane normal. At 30°C, the peptide undergoes intermediate time scale motions.


  1. Afonin S, Durr UH, Glaser RW, Ulrich AS (2004) ‘Boomerang’-like insertion of a fusogenic peptide in a lipid membrane revealed by solid-state 19F NMR. Magn Reson Chem 42:195–203CrossRefGoogle Scholar
  2. Andronesi OC, Becker S, Seidel K, Heise H, Young HS, Baldus M (2005) Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy. J Am Chem Soc 127:12965–12974CrossRefGoogle Scholar
  3. Barré P, Zschörnig O, Arnold K, Huster D (2003) Structural and dynamical changes of the bindin B18 peptide upon binding to lipid membranes. A solid-state NMR study. Biochemistry 42:8377–8386CrossRefGoogle Scholar
  4. Bechinger B (1999) The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy. Biochim Biophys Acta 1462:157–183CrossRefGoogle Scholar
  5. Bechinger B, Aisenbrey C, Bertani P (2004) The alignment, structure and dynamics of membrane-associated polypeptides by solid-state NMR spectroscopy. Biochim Biophys Acta 1666:190–204Google Scholar
  6. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG (1995) Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958CrossRefADSGoogle Scholar
  7. Berczi A, Horvath G (2003) Lipid rafts in the plant plasma membrane? Acta Biol Szeged 47:7–10Google Scholar
  8. Casey PJ (1995) Protein lipidation in cell signaling. Science 268:221–225CrossRefADSGoogle Scholar
  9. Davis JH, Auger M (1999) Static and magic angle spinning NMR of membrane peptides and proteins. Prog Nucl Magn Reson Spectrosc 35:1–84CrossRefGoogle Scholar
  10. Davis JH, Jeffrey KR, Bloom M, Valic MI, Higgs TP (1976) Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem Phys Lett 42:390–394CrossRefADSGoogle Scholar
  11. Eckhoff A, Granzin J, Kamphausen T, Büldt G, Schulz B, Weiergraber OH (2005) Crystallization and preliminary X-ray analysis of immunophilin-like FKBP42 from Arabidopsis thaliana. Acta Crystallograph Sect F Struct Biol Cryst Commun 61:363–365CrossRefGoogle Scholar
  12. Eisenberg D, Weiss RM, Terwilliger TC (1982) The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature 299:371–374CrossRefADSGoogle Scholar
  13. Eisenberg D, Weiss RM, Terwilliger TC (1984) The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Natl Acad Sci USA 81:140–144CrossRefADSGoogle Scholar
  14. Eisenberg D, Wesson M, Wilcox W (1989) Hydrophobic moments as tools for analyzing protein sequences and structures. In: Fasman GD (ed) Prediction of protein structure and the principles of protein conformation. Plenum Press, New York, London, pp 635–646Google Scholar
  15. Geisler M, Murphy AS (2006) The ABC of auxin transport: the role of p-glycoproteins in plant development. FEBS Lett 580:1094–1102CrossRefGoogle Scholar
  16. Geisler M, Kolukisaoglu HU, Bouchard R, Billion K, Berger J, Saal B, Frangne N, Koncz-Kalman Z, Koncz C, Dudler R, Blakeslee JJ, Murphy AS, Martinoia E, Schulz B (2003) TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19. Mol Biol Cell 14:4238–4249CrossRefGoogle Scholar
  17. Geisler M, Girin M, Brandt S, Vincenzetti V, Plaza S, Paris N, Kobae Y, Maeshima M, Billion K, Kolukisaoglu UH, Schulz B, Martinoia E (2004) Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters. Mol Biol Cell 15:3393–3405CrossRefGoogle Scholar
  18. Hahn EL (1950) Spin echoes. Phys Rev 80:580–594MATHCrossRefADSGoogle Scholar
  19. Hancock JF, Cadwallader K, Paterson H, Marshall CJ (1991) A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J 10:4033–4039Google Scholar
  20. Hashimoto Y, Toma K, Nishikido J, Yamamoto K, Haneda K, Inazu T, Valentine KG, Opella SJ (1999) Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy. Biochemistry 38:8377–8384CrossRefGoogle Scholar
  21. Henzler-Wildman KA, Lee DK, Ramamoorthy A (2003) Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37. Biochemistry 42:6545–6558CrossRefGoogle Scholar
  22. Henzler-Wildman KA, Martinez GV, Brown MF, Ramamoorthy A (2004) Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37. Biochemistry 43:8459–8469CrossRefGoogle Scholar
  23. Hessa T, Kim H, Bihlmaier K, Lundin C, Boekel J, Andersson H, Nilsson I, White SH, von Heijne G (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433:377–381CrossRefADSGoogle Scholar
  24. Hohwy M, Rienstra CM, Jaroniec CP, Griffin RG (1999) Fivefold symmetric homonuclear dipolar recoupling in rotating solids: application to double quantum spectroscopy. J Chem Phys 110:7983–7992CrossRefADSGoogle Scholar
  25. Huster D (2005) Investigations of the structure and dynamics of membrane-associated peptides by magic angle spinning NMR. Prog Nucl Magn Reson Spectrosc 46:79–107CrossRefGoogle Scholar
  26. Huster D, Arnold K, Gawrisch K (1998) Influence of docosahexaenoic acid and cholesterol on lateral lipid organization in phospholipid mixtures. Biochemistry 37:17299–17308CrossRefGoogle Scholar
  27. Huster D, Yao X, Jakes KS, Hong M (2002) Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study. Biochim Biophys Acta 1561:159–170CrossRefGoogle Scholar
  28. Huster D, Vogel A, Katzka C, Scheidt HA, Binder H, Dante S, Gutberlet T, Zschörnig O, Waldmann H, Arnold K (2003) Membrane insertion of a lipidated ras peptide studied by FTIR, solid-state NMR, and neutron diffraction spectroscopy. J Am Chem Soc 125:4070–4079CrossRefGoogle Scholar
  29. Igumenova TI, Wand AJ, McDermott AE (2004) Assignment of the backbone resonances for microcrystalline ubiquitin. J Am Chem Soc 126:5323–5331CrossRefGoogle Scholar
  30. Ikezawa H (2002) Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol Pharm Bull 25:409–417CrossRefGoogle Scholar
  31. Jayasinghe S, Hristova K, White SH (2001) Energetics, stability, and prediction of transmembrane helices. J Mol Biol 312:927–934CrossRefGoogle Scholar
  32. Kamphausen T, Fanghanel J, Neumann D, Schulz B, Rahfeld JU (2002) Characterization of Arabidopsis thaliana AtFKBP42 that is membrane-bound and interacts with Hsp90. Plant J 32:263–276CrossRefGoogle Scholar
  33. Ketchem RR, Hu W, Cross TA (1993) High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR. Science 261:1457–1460CrossRefADSGoogle Scholar
  34. Killian JA (1998) Hydrophobic mismatch between proteins and lipids in membranes. Biochim Biophys Acta 1376:401–415Google Scholar
  35. Koenig BW, Ferretti JA, Gawrisch K (1999) Site-specific deuterium order parameters and membrane-bound behavior of a peptide fragment from the intracellular domain of HIV-1 gp41. Biochemistry 38:6327–6334CrossRefGoogle Scholar
  36. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580CrossRefGoogle Scholar
  37. Ladokhin AS, White SH (1999) Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin. J Mol Biol 285:1363–1369CrossRefGoogle Scholar
  38. Lafleur M, Fine B, Sternin E, Cullis PR, Bloom M (1989) Smoothed orientational order profile of lipid bilayers by 2H-nuclear magnetic resonance. Biophys J 56:1037–1041CrossRefGoogle Scholar
  39. Lee DK, Santos JS, Ramamoorthy A (1999) Application of one-dimensional dipolar shift solid-state NMR spectroscopy to study the backbone conformation of membrane-associated peptides in phospholipid bilayers. J Phys Chem B 103:8383–8390CrossRefGoogle Scholar
  40. Luca S, Filippov DV, van Boom JH, Oschkinat H, de Groot HJ, Baldus M (2001) Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning. J Biomol NMR 20:325–331CrossRefGoogle Scholar
  41. Luca S, Heise H, Baldus M (2003) High-resolution solid-state NMR applied to polypeptides and membrane proteins. Acc Chem Res 36:858–865CrossRefGoogle Scholar
  42. Marassi FM, Ma C, Gratkowski H, Straus SK, Strebel K, Oblatt-Montal M, Montal M, Opella SJ (1999) Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1. Proc Natl Acad Sci USA 96:14336–14341CrossRefADSGoogle Scholar
  43. Marshall CJ (1993) Protein prenylation: a mediator of protein–protein interactions. Science 259:1865–1866CrossRefADSGoogle Scholar
  44. Mecke A, Lee DK, Ramamoorthy A, Orr BG, Banaszak Holl MM (2005) Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers. Biophys J 89:4043–4050CrossRefGoogle Scholar
  45. Morcombe CR, Zilm KW (2003) Chemical shift referencing in MAS solid state NMR. J Magn Reson 162:479–486CrossRefADSGoogle Scholar
  46. Mouritsen OG, Bloom M (1984) Mattress model of lipid–protein interactions in membranes. Biophys J 46:141–153Google Scholar
  47. Murray D, Ben-Tal N, Honig B, McLaughlin S (1997) Electrostatic interaction of myristoylated proteins with membranes: simple physics, complicated biology. Structure 5:985–989CrossRefGoogle Scholar
  48. Opella SJ, Marassi FM (2004) Structure determination of membrane proteins by NMR spectroscopy. Chem Rev 104:3587–3606CrossRefGoogle Scholar
  49. Porcelli F, Buck-Koehntop BA, Thennarasu S, Ramamoorthy A, Veglia G (2006) Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy. Biochemistry 45:5793–5799CrossRefGoogle Scholar
  50. Ramamoorthy A, Wei YF, Lee DK (2004) PISEMA solid-state NMR spectroscopy. Ann Rep NMR Spectrosc 52:1–52CrossRefGoogle Scholar
  51. Reuther G, Tan K-T, Köhler J, Nowak C, Pampel A, Arnold K, Kuhlmann J, Waldmann H, Huster D (2006) Structural model of the membrane-bound C terminus of lipid-modified human N-ras protein. Angew Chem Int Ed Engl 45:5387–5390CrossRefGoogle Scholar
  52. Saitô H (1986) Conformation-dependent 13C chemical shifts: a new means of conformational characterization as obtained by high-resolution solid-state 13C NMR. Magn Reson Chem 24:835–852CrossRefGoogle Scholar
  53. Saitô H, Tuzi S, Yamaguchi S, Tanio M, Naito A (2000) Conformation and backbone dynamics of bacteriorhodopsin revealed by 13C-NMR. Biochim Biophys Acta 1460:39–48CrossRefGoogle Scholar
  54. Schiffer M, Chang CH, Stevens FJ (1992) The functions of tryptophan residues in membrane proteins. Protein Eng 5:213–214CrossRefGoogle Scholar
  55. Seul M, Sammon MJ (1990) Preparation of urfactant multilayer films on solid substrates by deposition from organic solution. Thin Solid Films 185:287–305CrossRefADSGoogle Scholar
  56. Sharpe S, Yau WM, Tycko R (2006) Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid-state NMR with magic-angle spinning. Biochemistry 45:918–933CrossRefGoogle Scholar
  57. Smith SO, Palings I, Copie V, Raleigh DP, Courtin J, Pardoen JA, Lugtenburg J, Mathies RA, Griffin RG (1987) Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin. Biochemistry 26:1606–1611CrossRefGoogle Scholar
  58. Smith SO, Song D, Shekar S, Groesbeek M, Ziliox M, Aimoto S (2001) Structure of the transmembrane dimer interface of glycophorin A in membrane bilayers. Biochemistry 40:6553–6558CrossRefGoogle Scholar
  59. Spera S, Bax A (1991) Empirical correlation between protein backbone conformation and Cα and Cβ 13C nuclear magnetic resonance chemical shifts. J Am Chem Soc 113:5490–5492CrossRefGoogle Scholar
  60. Szeverenyi NM, Sullivan MJ, Maciel GE (1982) Observation of spin exchange by two-dimensional Fourier transform 13C cross polarization magic-angle spinning. J Magn Reson 47:462–475Google Scholar
  61. Thompson LK (2002) Solid-state NMR studies of the structure and mechanisms of proteins. Curr Opin Struct Biol 12:661–669CrossRefGoogle Scholar
  62. Torres J, Stevens TJ, Samso M (2003) Membrane proteins: the ‘Wild West’ of structural biology. Trends Biochem Sci 28:137–144CrossRefGoogle Scholar
  63. Uemura M, Joseph RA, Steponkus PL (1995) Cold Acclimation of Arabidopsis thaliana (effect on plasma membrane lipid composition and freeze-induced lesions). Plant Physiol 109:15–30Google Scholar
  64. Ulmschneider MB, Sansom MS, Di NA (2005) Properties of integral membrane protein structures: derivation of an implicit membrane potential. Proteins 59:252–265CrossRefGoogle Scholar
  65. Wagner K, Beck-Sickinger AG, Huster D (2004) Structural investigations of a human calcitonin-derived carrier peptide in membrane environment by solid-state NMR. Biochemistry 43:12459–81246CrossRefGoogle Scholar
  66. Wallin E, Tsukihara T, Yoshikawa S, von Heijne G, Elofsson A (1997) Architecture of helix bundle membrane proteins: an analysis of cytochrome c oxidase from bovine mitochondria. Protein Sci 6:808–815CrossRefGoogle Scholar
  67. Warschawski DE, Gross JD, Griffin RG (1998) Effects of membrane peptide dynamics on high-resolution magic-angle spinning NMR. J Chim Phys 95:460–466CrossRefGoogle Scholar
  68. Wasniewski CM, Parkanzky PD, Bodner ML, Weliky DP (2004) Solid-state nuclear magnetic resonance studies of HIV and influenza fusion peptide orientations in membrane bilayers using stacked glass plate samples. Chem Phys Lipids 132:89–100CrossRefGoogle Scholar
  69. Weiergraber OH, Eckhoff A, Granzin J (2006) Crystal structure of a plant immunophilin domain involved in regulation of MDR-type ABC transporters. FEBS Lett 580:251–255CrossRefGoogle Scholar
  70. White SH, von Heijne G (2005) Transmembrane helices before, during, and after insertion. Curr Opin Struct Biol 15:378–386CrossRefGoogle Scholar
  71. White SH, Wimley WC (1998) Hydrophobic interactions of peptides with membrane interfaces. Biochim Biophys Acta 1376:339–352Google Scholar
  72. White SH, Wimley WC (1999) Membrane protein folding and stability: physical principles. Annu Rev Biophys Biomol Struct 28:319–365CrossRefGoogle Scholar
  73. Wimley WC, White SH (1996) Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat Struct Biol 3:842–848CrossRefGoogle Scholar
  74. Wishart DS, Sykes BD (1994) Chemical shifts as a tool for structure determination. Methods Enzymol 239:363–392CrossRefGoogle Scholar
  75. Yang J, Weliky DP (2003) Solid-state nuclear magnetic resonance evidence for parallel and antiparallel strand arrangements in the membrane-associated HIV-1 fusion peptide. Biochemistry 42:11879–11890CrossRefGoogle Scholar
  76. Yau WM, Wimley WC, Gawrisch K, White SH (1998) The preference of tryptophan for membrane interfaces. Biochemistry 37:14713–14718CrossRefGoogle Scholar
  77. Zhang W, Crocker E, McLaughlin S, Smith SO (2003) Binding of peptides with basic and aromatic residues to bilayer membranes: phenylalanine in the MARCKS effector domain penetrates into the hydrophobic core of the bilayer. J Biol Chem 278:21459–21466CrossRefGoogle Scholar

Copyright information

© EBSA 2006

Authors and Affiliations

  • Holger A. Scheidt
    • 1
  • Alexander Vogel
    • 1
  • Andreas Eckhoff
    • 2
  • Bernd W. Koenig
    • 2
    • 3
  • Daniel Huster
    • 1
  1. 1.Junior Research Group “Structural Biology of Membrane Proteins”, Institute of BiotechnologyMartin Luther University Halle-WittenbergHalleGermany
  2. 2.Structural Biology Institute, IBI-2Research Centre JülichJülichGermany
  3. 3.Physical Biology InstituteHeinrich Heine University DüsseldorfDusseldorfGermany

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