Journal of Biomolecular NMR

, Volume 55, Issue 4, pp 369–377 | Cite as

Modulating alignment of membrane proteins in liquid-crystalline and oriented gel media by changing the size and charge of phospholipid bicelles

  • Justin L. Lorieau
  • Alexander S. Maltsev
  • John M. Louis
  • Ad BaxEmail author


We demonstrate that alignment of a structured peptide or small protein solubilized in mixed phospholipid:detergent micelles or bicelles, when embedded in a compressed gel or liquid crystalline medium, can be altered by either changing the phospholipid aggregate shape, charge, or both together. For the hemagglutinin fusion peptide solubilized in bicelles, we show that bicelle shape and charge do not change its helical hairpin structure but impact its alignment relative to the alignment medium, both in charged compressed acrylamide gel and in liquid crystalline d(GpG). The method can be used to generate sets of residual dipolar couplings that correspond to orthogonal alignment tensors, and holds promise for high-resolution structural refinement and dynamic mapping of membrane proteins.


Bicelle Dipolar coupling Fusion peptide NMR Orthogonal alignment Saupe matrix 



We thank Annie Aniana for help with protein expression and purification, Nicolas A. Bax for measuring the cmc of DHPS, and Dennis A. Torchia for discussions and comments. This work was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH) and the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH.

Supplementary material

10858_2013_9720_MOESM1_ESM.pdf (201 kb)
Supplementary material 1 (PDF 201 kb)


  1. Al-Hashimi HM, Valafar H, Terrell M, Zartler ER, Eidsness MK, Prestegard JH (2000) Variation of molecular alignment as a means of resolving orientational ambiguities in protein structures from dipolar couplings. J Magn Reson 143:402–406CrossRefADSGoogle Scholar
  2. Bax A (2003) Weak alignment offers new NMR opportunities to study protein structure and dynamics. Protein Sci 12:1–16CrossRefGoogle Scholar
  3. Bax A, Kontaxis G, Tjandra N (2001) Dipolar couplings in macromolecular structure determination. Meth Enzymol 339:127–174CrossRefGoogle Scholar
  4. Bertini I, Del Bianco C, Gelis I, Katsaros N, Luchinat C, Parigi G, Peana M, Provenzani A, Zoroddu MA (2004) Experimentally exploring the conformational space sampled by domain reorientation in calmodulin. Proc Natl Acad Sci USA 101:6841–6846CrossRefADSGoogle Scholar
  5. Bertini I, Kursula P, Luchinat C, Parigi G, Vahokoski J, Wilmanns M, Yuan J (2009) Accurate solution structures of proteins from X-ray data and a minimal set of NMR data: calmodulin-peptide complexes as examples. J Am Chem Soc 131:5134–5144CrossRefGoogle Scholar
  6. Blackledge M (2005) Recent progress in the study of biomolecular structure and dynamics in solution from residual dipolar couplings. Prog Nucl Magn Reson Spectrosc 46:23–61CrossRefGoogle Scholar
  7. Briggman KB, Tolman JR (2003) De Novo determination of bond orientations and order parameters from residual dipolar couplings with high accuracy. J Am Chem Soc 125:10164–10165CrossRefGoogle Scholar
  8. Cantor CR, Schimmel PR (1980) Biophysical chemistry. Freeman, San FranciscoGoogle Scholar
  9. Chou JJ, Gaemers S, Howder B, Louis JM, Bax A (2001) A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles. J Biomol NMR 21:377–382CrossRefGoogle Scholar
  10. Chou JJ, Kaufman JD, Stahl SJ, Wingfield PT, Bax A (2002) Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipolar coupling measurement in a stretched polyacrylamide gel. J Am Chem Soc 124:2450–2451CrossRefGoogle Scholar
  11. Chou JJ, Baber JL, Bax A (2004) Characterization of phospholipid mixed micelles by translational diffusion. J Biomol NMR 29:299–308CrossRefGoogle Scholar
  12. Cierpicki T, Bushweller JH (2004) Charged gels as orienting media for measurement of residual dipolar couplings in soluble and integral membrane proteins. J Am Chem Soc 126:16259–16266CrossRefGoogle Scholar
  13. Clore GM, Garrett DS (1999) R-factor, free R, and complete cross-validation for dipolar coupling refinement of NMR structures. J Am Chem Soc 121:9008–9012CrossRefGoogle Scholar
  14. Clore GM, Starich MR, Gronenborn AM (1998) Measurement of residual dipolar couplings of marcomolecules aligned in the nematic phase of a colloidal suspension of rod-shaped viruses. J Am Chem Soc 120:10571–10572CrossRefGoogle Scholar
  15. Clore GM, Starich MR, Bewley CA, Cai ML, Kuszewski J (1999) Impact of residual dipolar couplings on the accuracy of NMR structures determined from a minimal number of NOE restraints. J Am Chem Soc 121:6513–6514CrossRefGoogle Scholar
  16. Cornilescu G, Marquardt JL, Ottiger M, Bax A (1998) Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J Am Chem Soc 120:6836–6837CrossRefGoogle Scholar
  17. De Angelis AA, Opella SJ (2007) Bicelle samples for solid-state NMR of membrane proteins. Nat Protoc 2:2332–2338CrossRefGoogle Scholar
  18. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRpipe: a multidimensional spectral processing system based on Unix pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  19. Douglas SM, Chou JJ, Shih WM (2007) DNA-nanotube-induced alignment of membrane proteins for NMR structure determination. Proc Natl Acad Sci USA 104:6644–6648CrossRefADSGoogle Scholar
  20. Gaemers S, Bax A (2001) Morphology of three lyotropic liquid crystalline biological NMR media studied by translational diffusion anisotropy. J Am Chem Soc 123:12343–12352CrossRefGoogle Scholar
  21. Ghana R, Walss C, Walmsley JA (1996) Sodium and potassium ion-promoted formation of supramolecular aggregates of 2′-deoxyguanylyl-(3′-5′)-2′-deoxyguanosine. J Biomol Struct Dyn 14:101–110CrossRefGoogle Scholar
  22. Goddard TD, Kneller DG (2008) Sparky 3. University of California, San FranciscoGoogle Scholar
  23. Han X, Tamm LK (2000) A host-guest system to study structure-function relationships of membrane fusion peptides. Proc Natl Acad Sci USA 97:13097–13102CrossRefADSGoogle Scholar
  24. Hansen MR, Mueller L, Pardi A (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nature Struct Biol 5:1065–1074CrossRefGoogle Scholar
  25. Hus JC, Peti W, Griesinger C, Bruschweiler R (2003) Self-consistency analysis of dipolar couplings in multiple alignments of ubiquitin. J Am Chem Soc 125:5596–5597CrossRefGoogle Scholar
  26. Hus J-C, Salmon L, Bouvignies G, Lotze J, Blackledge M, Brueschweiler R (2008) 16-fold degeneracy of peptide plane orientations from residual dipolar couplings: analytical treatment and implications for protein structure determination. J Am Chem Soc 130:15927–15937CrossRefGoogle Scholar
  27. Kamen DE, Cahill SM, Girvin ME (2007) Multiple alignment of membrane proteins for measuring residual dipolar couplings using lanthanide ions bound to a small metal chelator. J Am Chem Soc 129:1846–1847CrossRefGoogle Scholar
  28. Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665CrossRefGoogle Scholar
  29. Lorieau J, Yao LS, Bax A (2008) Liquid crystalline phase of G-tetrad DNA for NMR study of detergent-solubilized proteins. J Am Chem Soc 130:7536–7537CrossRefGoogle Scholar
  30. Lorieau JL, Louis JM, Bax A (2010) The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid: water interface. Proc Natl Acad Sci USA 107:11341–11346CrossRefADSGoogle Scholar
  31. Lorieau JL, Louis JM, Bax A (2011) Whole-body rocking motion of a fusion peptide in lipid bilayers from size-dispersed (15)N NMR relaxation. J Am Chem Soc 133:14184–14187CrossRefGoogle Scholar
  32. Losonczi JA, Andrec M, Fischer MWF, Prestegard JH (1999) Order matrix analysis of residual dipolar couplings using singular value decomposition. J Magn Reson 138:334–342CrossRefADSGoogle Scholar
  33. Meier S, Haussinger D, Grzesiek S (2002) Charged acrylamide copolymer gels as media for weak alignment. J Biomol NMR 24:351–356CrossRefGoogle Scholar
  34. Meiler J, Prompers JJ, Peti W, Griesinger C, Bruschweiler R (2001) Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins. J Am Chem Soc 123:6098–6107CrossRefGoogle Scholar
  35. Mueller GA, Choy WY, Yang DW, Forman-Kay JD, Venters RA, Kay LE (2000) Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein. J Mol Biol 300:197–212CrossRefGoogle Scholar
  36. Ottiger M, Bax A (1999) Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values. J Biomol NMR 13:187–191CrossRefGoogle Scholar
  37. Peti W, Meiler J, Bruschweiler R, Griesinger C (2002) Model-free analysis of protein backbone motion from residual dipolar couplings. J Am Chem Soc 124:5822–5833CrossRefGoogle Scholar
  38. Prestegard JH, Al-Hashimi HM, Tolman JR (2000) NMR structures of biomolecules using field oriented media and residual dipolar couplings. Q Rev Biophys 33:371–424CrossRefGoogle Scholar
  39. Ramirez BE, Bax A (1998) Modulation of the alignment tensor of macromolecules dissolved in a dilute liquid crystalline medium. J Am Chem Soc 120:9106–9107CrossRefGoogle Scholar
  40. Rodriguez-Castaneda F, Haberz P, Leonov A, Griesinger C (2006) Paramagnetic tagging of diamagnetic proteins for solution NMR. Magn Reson Chem 44:S10–S16CrossRefGoogle Scholar
  41. Ruan K, Tolman JR (2005) Composite alignment media for the measurement of independent sets of NMR residual dipolar couplings. J Am Chem Soc 127:15032–15033CrossRefGoogle Scholar
  42. Ruan K, Briggman KB, Tolman JR (2008) De novo determination of internuclear vector orientations from residual dipolar couplings measured in three independent alignment media. J Biomol NMR 41:61–76CrossRefGoogle Scholar
  43. Ruckert M, Otting G (2000) Alignment of biological macromolecules in novel nonionic liquid crystalline media for NMR experiments. J Am Chem Soc 122:7793–7797CrossRefGoogle Scholar
  44. Sanders CR, Schwonek JP (1992) Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR. Biochemistry 31:8898–8905CrossRefGoogle Scholar
  45. Sass J, Cordier F, Hoffmann A, Rogowski M, Cousin A, Omichinski JG, Lowen H, Grzesiek S (1999) Purple membrane induced alignment of biological macromolecules in the magnetic field. J Am Chem Soc 121:2047–2055CrossRefGoogle Scholar
  46. Sass H-J, Musco G, Stahl SJ, Wingfield PT, Grzesiek S (2000) Solution NMR of proteins within polyacrylamide gels: diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes. J Biomol NMR 18:303–309CrossRefGoogle Scholar
  47. Saupe A, Englert G (1963) High-resolution nuclear magnetic resonance spectra of oriented molecules. Phys Rev Lett 11:462–464CrossRefADSGoogle Scholar
  48. Shortle D, Ackerman MS (2001) Persistence of native-like topology in a denatured protein in 8 M urea. Science 293:487–489CrossRefGoogle Scholar
  49. Struppe J, Whiles JA, Vold RR (2000) Acidic phospholipid bicelles: a versatile model membrane system. Biophys J 78:281–289CrossRefGoogle Scholar
  50. Su XC, Otting G (2010) Paramagnetic labelling of proteins and oligonucleotides for NMR. J Biomol NMR 46:101–112CrossRefGoogle Scholar
  51. Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114CrossRefADSGoogle Scholar
  52. Tolman JR (2002) A novel approach to the retrieval of structural and dynamic information from residual dipolar couplings using several oriented media in biomolecular NMR spectroscopy. J Am Chem Soc 124:12020–12030CrossRefGoogle Scholar
  53. Tolman JR, Ruan K (2006) NMR residual dipolar couplings as probes of biomolecular dynamics. Chem Rev 106:1720–1736CrossRefGoogle Scholar
  54. Tolman JR, Flanagan JM, Kennedy MA, Prestegard JH (1995) Nuclear magnetic dipole interactions in field-oriented proteins—information for structure determination in solution. Proc Natl Acad Sci USA 92:9279–9283CrossRefADSGoogle Scholar
  55. Tycko R, Blanco FJ, Ishii Y (2000) Alignment of biopolymers in strained gels: a new way to create detectable dipole–dipole couplings in high-resolution biomolecular NMR. J Am Chem Soc 122:9340–9341CrossRefGoogle Scholar
  56. Ulmer TS, Ramirez BE, Delaglio F, Bax A (2003) Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy. J Am Chem Soc 125:9179–9191CrossRefGoogle Scholar
  57. Vold RR, Prosser RS (1996) Magnetically oriented phospholipid bilayered micelles for structural studies of polypeptides. Does the ideal bicelle exist? J Magn Reson, Ser B 113:267–271CrossRefGoogle Scholar
  58. Vold RR, Prosser RS, Deese AJ (1997) Isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution NMR studies of polypeptides. J Biomol NMR 9:329–335CrossRefGoogle Scholar
  59. Wohnert J, Franz KJ, Nitz M, Imperiali B, Schwalbe H (2003) Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings. J Am Chem Soc 125:13338–13339CrossRefGoogle Scholar
  60. Yao LS, Bax A (2007) Modulating protein alignment in a liquid-crystalline medium through conservative mutagenesis. J Am Chem Soc 129:11326–11327CrossRefGoogle Scholar
  61. Yao L, Vogeli B, Torchia DA, Bax A (2008) Simultaneous NMR study of protein structure and dynamics using conservative mutagenesis. J Phys Chem B 112:6045–6056CrossRefGoogle Scholar
  62. Yao LS, Ying JF, Bax A (2009) Improved accuracy of N-15-H-1 scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins. J Biomol NMR 43:161–170CrossRefGoogle Scholar
  63. Zhang Q, Sun XY, Watt ED, Al-Hashimi HM (2006) Resolving the motional modes that code for RNA adaptation. Science 311:653–656CrossRefADSGoogle Scholar
  64. Zidek L, Padrta P, Chmelik J, Sklenar V (2003) Internal consistency of NMR data obtained in partially aligned biomacromolecules. J Magn Reson 162:385–395CrossRefADSGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht (outside the USA) 2013

Authors and Affiliations

  • Justin L. Lorieau
    • 1
  • Alexander S. Maltsev
    • 1
  • John M. Louis
    • 1
  • Ad Bax
    • 1
    Email author
  1. 1.Laboratory of Chemical PhysicsNational Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of HealthBethesdaUSA

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