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Burkhard Bechinger Université Louis Pasteur, CNRS - UMR

Investigations of Membrane Polypeptides by Solid-state NMR Spectroscopy: Structure, Dynamics, Aggregation and Topology of Supramolecular Complexes. Burkhard Bechinger Université Louis Pasteur, CNRS - UMR Chimie-physique moléculaire et spectroscopie Strasbourg, France. Solution NMR

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Burkhard Bechinger Université Louis Pasteur, CNRS - UMR

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  1. Investigations of Membrane Polypeptides by Solid-state NMR Spectroscopy: Structure, Dynamics, Aggregation and Topology of Supramolecular Complexes Burkhard Bechinger Université Louis Pasteur, CNRS - UMR Chimie-physique moléculaire et spectroscopie Strasbourg, France

  2. Solution NMR Requires fast and isotropic motional averaging < 120 kDa (TROSY) Solid-state NMR frozen, dry liquid crystalline membranes no physial size limitation Structure, orientation and dynamics NMR to study membrane proteins

  3. Solution NMR Requires fast and isotropic motional averaging < 120 kDa (TROSY) Extended liquid crystalline bilayers are too big Solid-state NMR frozen, dry liquid crystalline membranes no physical size limitation Structure, orientation and dynamics NMR to study membrane proteins

  4. Solid-state NMR provides information on … • chemical environment • distances • dihedral angles • orientations in space Structure, dynamics and topology

  5. Oriented membranes Bo

  6. Chemical synthesis of peptides allows labelling at single sites

  7. Oriented Samples:Structure and Topology

  8. 15N chemical shift  alignment of the peptide bond

  9. Solution and solid-state NMR on the same scale

  10. Bo DnQ ~ 3 cos2Q-1 2H 2H 2H Cb Ca Q The 2H quadrupolar splitting2H3-alanine Similar principles apply for many NMR interactions

  11. 2 angles 2 measurables Detailed helix alignment from combined 15N and 2H measurements

  12. + + + + + Tilt 95o, pitch 173o Unique solution from Energy Minimization hydrophobic hydrophilic +

  13. Difference 2o KL14 Model Peptide in Oriented Phosphatidylcholine Bilayers Lipid 2H (kHz) 15N (ppm) POPC 6.0 74 DMPC 7.6 73 PC20:1 8.3 73 DOPC 10.8 74

  14. Dynamics:Rotational Diffusion and Aggregation

  15. Liquid crystalline membranes Motion around the membrane normal

  16. Rotational averaging:Effect on 15N powder pattern line shape Static Rotation around s33 (helix long axis) Rotation around s22 Powder pattern provide orientational information

  17. 2H solid-state NMR2H3-alanyl

  18. IP helix TM helix Loss of intensity during transition Freezing Rotational Diffusion

  19. 2H-NMR Equilibrium: Mono- / oligomer

  20. 2H NMR of ‘‘real‘‘ samplese.g. viral channel peptides Influenza M2 Vpu

  21. 2H spectral line shape and mosaic spread Tilt angle: 10o 40o 50o 700 Mosaic Spread 0.5 1 3 5 10 15

  22. Q= 45.3o or 65.5o Mosaic spread = 1o Model amphipathic helix

  23. Example:Controlling Topology

  24. Oriented 15N solid-state NMR:LAH4  pH-dependent molecular switch

  25. Example: Domain of ICP47 • Herpes simplex virus • 87 residues early gene product (domain 2-34 active) • Inhibits transport by TAP of antigenic peptides to surface and thus presentation by MHC I  lack of immunogenic response • Solution NMR: Helix (5-14)-loop-helix (22-31) in SDS micelles c/o Robert Tampé - Frankfurt

  26. tilt 84o ‚Modelling‘ tilt 75o 15N solid-state NMR of ICP47(2-34) in oriented POPC Helix1 Loop Helix2

  27. Mosaic spread 10-15o 2H solid-state NMR of ICP47(2-34) in oriented POPC

  28. Model for membrane-bound ICP47

  29. Christopher Aisenbrey Christina Sizun Bas Vogt Jesus Raya Gérard Nullans, ULP-INSERM Neurochimie Robert Tampé, Universität Frankfurt € ARC, ANRS, Vaincre la Mucoviscidose Region Alsace CNRS, Ministère, ACI Jeune Equipe Acknowledgements

  30. Combine MAS and • oriented samples Methods to orient lipid bilayers

  31. MAOSS at 10 kHz 31P NMR of oriented bilayers 10 kHz 565 Hz simulated

  32. MAS side band analysis provides orientational information

  33. b=10o s=25o 3.7o mosaic 20 % powder MAOSS of hydrophobic model peptide in phospholipid bilayer 15N NMR 31P NMR

  34. Summary • MAOSS with new sample set up  low or fast spinning • spinning side band analysis  tilt, mosaic spread and powder pattern contributions

  35. Model for membrane-bound ICP47

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