Exploiting spectral anisotropy in membrane studies

1. Exploiting spectral anisotropy in membrane studies Dr Philip Williamson May 2009

2. Overview • Anisotropic interactions present in solid-state NMR spectra of biological membranes • How to exploit anisotropy in powder samples to give structural and functional information • Methods for the preparation of macroscopically aligned membranes • Techniques to exploit oriented samples to provide structural/dynamic information

3. Introduction to anisotropic interaction

4. How do anisotropic interaction affect the NMR spectrum • Each molecular orientation gives rise to a difference resonance frequency • In powder we have the sum of all distributions • In the liquid state these anisotropic properties are averaged on the NMR timescale

5. Which interactions in NMR Isotropic Anisotropic

6. Chemical Shielding Anisotropy • Perturbation of the magnetic field due to interaction with surrounding electrons • Inherently asymmetric (e.g. electron distribution surrounding carbonyl group)

7. Describing interactions: tensors • Second rank tensors k i j k i j z szz syy sxx y x

8. Chemical Shielding Anisotropy • We can describe the perturbation of the main field (B0), by the second rank tensor, s. • The Hamiltonian which describes the interaction with the modified field is: Which can be written in a simplified form as:

9. Chemical Shielding Anisotropy Thus the chemical shielding Hamiltonian simplifies to: and the resonance frequency of the line is: Thus the resonance frequency is proportional to szz in the laboratory frame. However, s is usually defined in the principle axis system (PAS) not in the lab frame (LF). Therefore, we need to transform s from the PAS to LF.

10. Transformation matrix Can derive a rotation matrix which bring about the rotation described above: To determine s in the laboratory frame, need to apply to the chemical shielding tensor s in the principle axis system: This can be simplified to give general Hamiltonian for CSA in lab frame of:

11. Effect on resonance position d/2 d z szz =3000Hz • siso = 1/3(sxx+syy+szz) = 0Hz • = szz-siso = 3000 Hz • h = (syy-sxx)/d = 0.0 syy=-1500Hz y sxx =-1500Hz x

12. Powder Patterns • In powders we have a random distribution of molecular orientations. • Thus the lineshape is the weighted superposition of all the different orientations:

13. Dipolar Interaction Classical interpretation Classical interaction energy between two magnetic (dipole) moments when both are aligned with the magnetic field: Quantum mechanical where: • Symmetric second rank axially symmetric tensor. • Again we need to rotate from the PAS to LF to obtain resonance frequency. B0 m2 q m1

14. Orientation dependence of dipolar interaction Homo-nuclear Dipolar Hamiltonian: Hetero-nuclear Dipolar Hamiltonian: 1/2ddip 3/4ddip ddip=20 kHz

15. Quadrupolar Interaction (1) If the spin>1/2 (e.g. 2H, 14N ...), the nucleus contains an electronic quadrupole moment (Q). Electronic quadrupole moment interacts with surrounding electron cloud (electric field gradient(EFG), V). where: Provides: • A good reporter on the local electronic distribution about the nucleus (e.g. H-bonding status) • Due to large anisotropy, good reporter for orientation studies

16. Quadrupolar Interaction (2) To calculate the resonance frequency, we must transform from the PAS of the EFG to the laboratory frame. Retaining only the “secular terms” gives the following Hamiltonian in the LF: Powder spectrum of Ala-d3 dQ Orientation dependence of a single crystal of Ala-d3

17. Powder samples

18. Anisotropy in disordered samples • Changes in electrostatic environment • Changes in size of anisotropy (CSA, Dipolar couplings) • Typically studied under MAS • Changes in dynamics • Ligand binding sites • Protein/Peptide dynamics

19. Scaling of anisotropic interactions • Can use different motional models to study averaging of anisotropic interactions: • Multisite jump • Rotational diffusion .... Membrane normal Peptide long axis

20. 2H-NMR dynamic studies of acetylcholine salts BrAChBr AChCl AChClO4 Field (B0) C3’ C3 • Temperature dependent • Lineshapes dominated by motions about the C3 and C3’axis of rotation • Lineshape provide information about energy barriers associated with rotation

21. Dynamics of 2H-BrACh whilst resident in the binding site on the nAChR ACh Perchlorate Bound BrACh Membrane reorientation Backbone dynamics C3/C3’ Rotation Reduction in backbone dynamics C3 or C3’ rotation hindered C3 and C3’ rotation hindered Field (B0) C3’ Cys192/193 C3 Rotation of quaternary ammonium group hindered in the binding site

22. Structure of the TMD of the nAChR Ala8-D3 Leu11-C1 Gly15-N M3 M4 Gly23-C2 M2 M1 (Ortells, 1999)

23. Averaging of anisotropic interactions in DoMPC vesicles 13C1-Leu11 15N-Gly15 2H3-Ala8 MAS MAS Static Lb phase Static MAS Static La phase

24. Membrane normal Peptide long axis Structure from dynamics in non-oriented systems 13C1-Leu11 15N-Gly15

25. Secondary Structure of the M4-TMD • CD Spectroscopy indicates • Over 50% of residues in a a-helical conformation • Conformation preserved in TFE and lipid bicelles

26. Membrane protein dynamics: APP b a g b b a a amyloid a+b amyloid b g g • Changes in lipid composition: • Lipid metabolism (Chol/Sph) • Lipid oxidation • Level of saturation

27. Protease cleavage site accessibility 2.30-2.90nm  3.60nm

28. Lipid induced elevated b-amyloid levels Change in oligomeric state Increase in bilayer thickness Protection from a-secretase b-amyloid

29. Orienting Biological Membranes

30. Degree of orientation: mosaic spread n Mosaic spread • “Slow” variation of membrane normal with respect to director Degree of sample alignment • Extracted from experimental data Typically modelled • Distribution (different models) about bilayer normal Db

31. Mechanical orientation of synthetic lipid bilayers Lipid/Peptide samples prepared from: • Solvent (CH3OH/CHCl3) • Vesicle Suspension • Mixtures containing naphthalene Drying/Hydration • Under vacuum followed by rehydration • Equilibration at constant humidity Sealed in container for measurement by NMR (prevent dehydration)

32. Salt solutions for maintaining hydration

33. Mechanical orientation Oriented Bacteriorhodopsin Spectra Powder Bacteriorhodopsin Spectra Purple membranes • Resolved signals from 2 phosphate groups in PGP • Linebroadening dense packing of protein Prepared by slow buffer evaporation Mosaic spread ±10º

34. Magnetic alignment: diamagnetic anisotropy • Lipids possess negative diamagnetic anisotropic • Spontaneously align in magnetic field with chains perpendicular to applied field • In ensembles such as lipid bilayers energy exceeds thermal fluctuations and bilayers align • Causes deformation of vesicles, apparent in 31P spectra B0

35. Formation of bicelles Addition of surfactant (DHPC, CHAPS etc …) results in: • Under correct condition (hydration, T, etc) these form small discoidal objects (or extended perforated phases) • These spontaneously align in the magnetic field Below phase transition, mixed micellar B0 b Above phase transition, discoidal particles - bicelles n

36. 0 -2 -4 DoHPC -6 31P Chemical Shift (ppm) -8 +M4 -M4 -10 DoMPC -12 5 10 15 20 25 30 35 40 Temperature(ºC) Macroscopic orientation of the M4-TMD in DoMPC:DoHPC bicelles DoMPC DoHPC +M4 DoMPC • Positive diamagnetic anisotropy of protein does not perturb alignment • Lineshape analysis indicates a mosaic spread of <4º (limited by intrinsic linewidth) DoHPC

37. Flipping the bicelle: advantages for NMR Conventional Bicelles Parallel bicelles Bilayer normal parallel to field Full anisotropy (S=1.0) Uniaxial distribution Bilayer normal perpendicular to field • Anisotropy halved (S=-0.5) • No rotation leads to cylindrical distribution B0 B0

38. Flipping the bicelle DMPC DMPC/Tm3+=150 Require molecules in bilayer which possess a diamagnetic anisotropy • 1-napthol (first) • Transmembrane peptides (gramacidin) • Surface associated lanthanides Eu3+, Er3+, Tm3+, and Yb3+ • Chelating lipids containing lanthanides DHPC DMPC/Tm3+=40 DMPC DMPE-DTPA/Tm3+=1 DHPC DMPE-DTPA Prosser, 1998

39. Macroscopic orientation of native membranes • Samples spun onto iso-potential surface • Can be combined with drying of the sample followed by rehydration Oriented erythrocyte membranes imaged by electron-microscopy (Analytical Biochemistry, 1998)

40. n B0 n Macroscopic orientation of native membranes Native nAChR membrane, pelleted onto Mellanex sheet, 25000 rpm overnight, no drying (Analytical Biochemistry, 1998)

41. Applications of oriented samples

42. Effect on resonance position d/2 d z szz =3000Hz • siso = 1/3(sxx+syy+szz) = 0Hz • = szz-siso = 3000 Hz • h = (syy-sxx)/d = 0.0 syy=-1500Hz y sxx =-1500Hz x

43. Orientation 0° 90° Deuterium NMR to probe ligand orientation Field (B0) C3’ Cys192/193 C3

44. Orientation±5° Orientation±5° Mosaic Spread±5° Mosaic Spread±5° Oriented samples – ligand orientations B0 B0

45. Quaternary ammonium group is restricted in binding site Change in conformation? Interaction with binding site? The quaternary ammonium group lies at 42° with respect to the bilayer normal A structural and dynamic description of BrACh in the ligand binding site

46. Conformation of peptides/proteins Probing orientation with 2H-NMR: • Excellent sensitivity to orientation • Labelled site connects direct to peptide backbone Restrictions: • Restricted to analysis of alanine residues • Difficult to analyse multiple sites • Labelling typically by peptide-synthesis

47. Orientation constraints from multiply labelled proteins For proteins and peptides • Need resolution • Characterise backbone orientation Solution • Exploit 15N chemical shielding anisotropy • 1H-15N dipolar coupling • Characterise orientation of peptide plane

48. PISEMA spectra Polarization inversion spin exchange at the magic angle • 15N chemical shielding anisotropy • 15N-1H dipolar interaction Good scaling factor (0.82) and can be implemented in 3/4D experiments to improve resolution 35.5º -X (p/2)X tm Decouple 1H -Y Y+LG -Y-LG X X X -X

49. PISEMA spectra of Fd coat protein 35.5º -X (p/2)X tm Decouple 1H -Y Y+LG -Y-LG X X X -X

50. Tilt of helices from PISA wheels PISA Polarity Index Slant Angle Position of wheels in PISEMA spectra give orientation of helices in samples Amphipathic helix on bilayer surface TMD 30º with respect to bilayer