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. NMR to study membrane proteins 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

3. NMR to study membrane proteins 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

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

5. Oriented membranes BoBo

6. Chemical synthesis of peptides allows labelling at single sites

7. Oriented Samples: Structure and Topology

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

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

10. The 2 H quadrupolar splitting 2 H 3 -alanine CC CC 2H2H 2H2H 2H2H  Q ~ 3 cos 2  -1 BoBo  Similar principles apply for many NMR interactions

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

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

13. KL14 Model Peptide in Oriented Phosphatidylcholine Bilayers Lipid 2 H (kHz) 15 N (ppm) POPC 6.074 DMPC 7.673 PC20:1 8.373 DOPC 10.874 Difference 2 o

14. Dynamics: Rotational Diffusion and Aggregation

15. Liquid crystalline membranes Motion around the membrane normal

16. Rotational averaging: Effect on 15 N powder pattern line shape Static Rotation around  33 (helix long axis) Rotation around  22 Powder pattern provide orientational information

17. 2 H solid-state NMR 2 H 3 -alanyl

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

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

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

21. 2 H spectral line shape and mosaic spread Tilt angle: 10 o 40 o 50 o 70 0 Mosaic Spread 0.5 1 3 5 10 15

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

23. Example: Controlling Topology

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

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

28. Model for membrane-bound ICP47

29. Acknowledgements 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

31. Methods to orient lipid bilayers Combine MAS and oriented samples

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

33. MAS side band analysis provides orientational information

34. MAOSS of hydrophobic model peptide in phospholipid bilayer 3.7 o mosaic 20 % powder 15 N NMR 31 P NMR  =10 o  =25 o

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

36. Model for membrane-bound ICP47