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
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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

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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

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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.

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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

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Effect on resonance position
d/2
d

• siso 1/3(sxxsyyszz) 0Hz
• szz-siso 3000 Hz
• h (syy-sxx)/d 0.0

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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.

14
Orientation dependence of dipolar interaction

• Homo-nuclear Dipolar Hamiltonian

• Hetero-nuclear Dipolar Hamiltonian

1/2ddip
3/4ddip
ddip20 kHz
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Quadrupolar Interaction (1)

• If the spingt1/2 (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

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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
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Powder samples
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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

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Scaling of anisotropic interactions

• Can use different motional models to study
  averaging of anisotropic interactions
• Multisite jump
• Rotational diffusion ....

20
2H-NMR dynamic studies of acetylcholine salts
BrAChBr
AChCl
AChClO4

• Temperature dependent
• Lineshapes dominated by motions about the C3 and
  C3axis of rotation
• Lineshape provide information about energy
  barriers associated with rotation

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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
Rotation of quaternary ammonium group hindered in
the binding site
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Structure of the TMD of the nAChR
Ala8-D3
Leu11-C1
Gly15-N
M3
M4
Gly23-C2
M2
M1
(Ortells, 1999)
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Averaging of anisotropic interactions in DoMPC
vesicles
15N-Gly15
13C1-Leu11
2H3-Ala8
MAS
MAS
Static
Lb phase
Static
MAS
Static
La phase
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Structure from dynamics in non-oriented systems
13C1-Leu11
15N-Gly15
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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 ab
amyloid b
g
g

• Changes in lipid composition
• Lipid metabolism (Chol/Sph)
• Lipid oxidation
• Level of saturation

27
Protease cleavage site accessibility
3.60nm
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Lipid induced elevated b-amyloid levels
Change in oligomeric state
Increase in bilayer thickness
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Orienting Biological Membranes
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Degree of orientation mosaic spread

• 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
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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)

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Salt solutions for maintaining hydration
Saturated aqueous solution with considerable precipitates relative air humidity above the solution (at 20 C)
di-Sodium hydrogen phosphate Na2HPO4 x 12 H2O 95
Sodium carbonate Na2CO3 92
Zinc sulfate ZnSO4 x 7 H2O 90
Potassium chloride KCl 86
Ammonium sulfate (NH4)2SO4 80
Sodium chloride NaCl 76
Sodium nitrite NaNO2 65
Ammonium nitrate NH4NO3 63
Calcium nitrate Ca (NO3)2 x 4 H2O 55
Potassium carbonate K2CO3 45
Zinc nitrate Zn (NO3)2 x 6 H2O 42
Calcium chloride CaCl2 x 6 H2O 32 32
Lithium chloride LiCl x H2O 15 15
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Mechanical orientation

• Purple membranes
• Resolved signals from 2 phosphate groups in PGP
• Linebroadening dense packing of protein
• Prepared by slow buffer evaporation
• Mosaic spread 10º

Oriented Bacteriorhodopsin Spectra
Powder Bacteriorhodopsin Spectra
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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

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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
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Macroscopic orientation of the M4-TMD in
DoMPCDoHPC bicelles
DoMPC
DoHPC
M4
DoMPC

• Positive diamagnetic anisotropy of protein does
  not perturb alignment
• Lineshape analysis indicates a mosaic spread of
  lt4º (limited by intrinsic linewidth)

DoHPC
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Flipping the bicelle advantages for NMR

• Conventional Bicelles

• Parallel bicelles

• Bilayer normal perpendicular to field
• Anisotropy halved (S-0.5)
• No rotation leads to cylindrical distribution

• Bilayer normal parallel to field
• Full anisotropy (S1.0)
• Uniaxial distribution

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Flipping the bicelle
DMPC
DMPC/Tm3150

• 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/Tm340
DMPC
DMPE-DTPA/Tm31
DHPC
DMPE-DTPA
Prosser, 1998
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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)
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Macroscopic orientation of native membranes
Native nAChR membrane, pelleted onto Mellanex
sheet, 25000 rpm overnight, no drying (Analytical
Biochemistry, 1998)
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Applications of oriented samples
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Effect on resonance position
d/2
d

• siso 1/3(sxxsyyszz) 0Hz
• szz-siso 3000 Hz
• h (syy-sxx)/d 0.0

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Deuterium NMR to probe ligand orientation
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Oriented samples ligand orientations
B0
B0
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A structural and dynamic description of BrACh in
the ligand binding site

• 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

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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

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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

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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
YLG
-Y-LG
X
X
X
-X
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PISEMA spectra of Fd coat protein
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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
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Assignment of PISEMA spectra
PISEMA Spectra of amino acid selectively labelled
Fd cost protein (Marrassi, 2002)
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Extracting structure dipolar waves

• Dipolar waves
• dipolar coupling verses residue
• periodicity arises from repeating structure
  (e.g. a-helix)
• enables comparisons to be made with rdcs in
  solution
• disruption in ideal nature of secondary structure
  readily apparent

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Dipolar waves Fd coat protein

• Breaks in wave indicate
• Start of new secondary structure
• Deformation in secondary structure (kinks in
  helices)

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Summary

• 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

55
References

• Anisotropic interactions
• Principles of NMR in one and two dimensions,
  Ernst, Bodenhausen Wokenau
• Averaging of anisotropic interaction
• Principles of Magnetic Resonance, C.P. Schlicter
• Orienting of biological membranes
• Marcotte I, Auger M. 2005 Bicelles as model
  membranes for solid- and solution-state NMR
  studies of membrane peptides and proteins.
  Concepts in Magnetic Resonance Part
  A24A(1)17-37.
• Triba MN, Zoonens M, Popot JL, Devaux PF,
  Warschawski DE. 2006 Reconstitution and alignment
  by a magnetic field of a beta-barrel membrane
  protein in bicelles. European Biophysics
  Journal35(3)268-275.
• Grobner G, Taylor A, Williamson PTF, Choi G,
  Glaubitz C, Watts JA, deGrip WJ, Watts A. 1997
  Macroscopic orientation of natural and model
  membranes for structural studies. Analytical
  Biochemistry254(1)132-138. (and references
  therein)
• Prosser RS, Hwang JS, Vold RR. 1998 Magnetically
  aligned phospholipid bilayers with positive
  ordering A new model membrane system.
  Biophysical Journal74(5)2405-2418.
• NMR studies of oriented biological membranes
• Ramamoorthy A, Wu CH, Opella SJ. 1999
  Experimental aspects of multidimensional
  solid-state NMR correlation spectroscopy. Journal
  of Magnetic Resonance140(1)131-140.
• Marassi FM, Opella SJ. 2003 Simultaneous
  assignment and structure determination of a
  membrane protein from NMR orientational
  restraints. Protein Science12(3)403-411.
• Kim S, Cross TA. 2004 2D solid state NMR spectral
  simulation of 3(10), alpha, and pi-helices.
  Journal of Magnetic Resonance168(2)187-193.
• Mesleh MF, Lee S, Veglia G, Thiriot DS, Marassi
  FM, Opella SJ. 2003 Dipolar waves map the
  structure and topology of helices in membrane
  proteins. Journal of the American Chemical
  Society1258928-8935.
• Mesleh MF, Opella SJ. 2003 Dipolar Waves as NMR
  maps of helices in proteins. Journal of Magnetic
  Resonance163(2)288-299.

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Acknowledgements

• University of Southampton
• School of Biological Sciences
• Dr Phedra Marius
• Garrick Taylor
• Phillippa Hunnisett
• Sarah Stephens
• Maiwenn Beaugrand
• Dr Jörn Werner
• Werner group
• Zara Luedke
• Dr Vincent OConnor
• Prof. Lindy Holden Dye
• Prof. Robert Walker
• School of Chemistry

• University of Southampton
• School of Engineering and Computing Science
• Dr Maurits dePlanque
• University College London
• Prof. Steve Wood
• ETH, Zurich
• Prof. Beat Meier
• Dr Aswin Verhoeven
• Dr Giorgia Zandomeneghi
• Meier Group
• Dr Stefanie Krämer
• Dr Marco Marenchino
• University of Oxford
• Prof. Tony Watts

• Funding