Title: Exploiting spectral anisotropy in membrane studies
1Exploiting spectral anisotropy in membrane studies
Dr Philip Williamson May 2009
2Overview
- 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
3Introduction to anisotropic interaction
4How 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
5Which interactions in NMR
Isotropic
Anisotropic
6Chemical Shielding Anisotropy
- Perturbation of the magnetic field due to
interaction with surrounding electrons - Inherently asymmetric (e.g. electron distribution
surrounding carbonyl group)
7Describing interactions tensors
8Chemical 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
-
9Chemical 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. -
10Transformation 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 -
11Effect on resonance position
d/2
d
- siso 1/3(sxxsyyszz) 0Hz
- szz-siso 3000 Hz
- h (syy-sxx)/d 0.0
12Powder Patterns
- In powders we have a random distribution of
molecular orientations. - Thus the lineshape is the weighted superposition
of all the different orientations
13Dipolar 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.
14Orientation dependence of dipolar interaction
- Homo-nuclear Dipolar Hamiltonian
- Hetero-nuclear Dipolar Hamiltonian
1/2ddip
3/4ddip
ddip20 kHz
15Quadrupolar 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
16Quadrupolar 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
17Powder samples
18Anisotropy 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
19Scaling of anisotropic interactions
- Can use different motional models to study
averaging of anisotropic interactions - Multisite jump
- Rotational diffusion ....
202H-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
21Dynamics 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
22Structure of the TMD of the nAChR
Ala8-D3
Leu11-C1
Gly15-N
M3
M4
Gly23-C2
M2
M1
(Ortells, 1999)
23Averaging of anisotropic interactions in DoMPC
vesicles
15N-Gly15
13C1-Leu11
2H3-Ala8
MAS
MAS
Static
Lb phase
Static
MAS
Static
La phase
24Structure from dynamics in non-oriented systems
13C1-Leu11
15N-Gly15
25Secondary Structure of the M4-TMD
- CD Spectroscopy indicates
- Over 50 of residues in a a-helical conformation
- Conformation preserved in TFE and lipid bicelles
26Membrane 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
27Protease cleavage site accessibility
3.60nm
28Lipid induced elevated b-amyloid levels
Change in oligomeric state
Increase in bilayer thickness
29Orienting Biological Membranes
30Degree 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
31Mechanical 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)
32Salt 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
33Mechanical 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
34Magnetic 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 -
35Formation 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
36Macroscopic 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
37Flipping the bicelle advantages for NMR
- 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
38Flipping 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
39Macroscopic 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)
40Macroscopic orientation of native membranes
Native nAChR membrane, pelleted onto Mellanex
sheet, 25000 rpm overnight, no drying (Analytical
Biochemistry, 1998)
41Applications of oriented samples
42Effect on resonance position
d/2
d
- siso 1/3(sxxsyyszz) 0Hz
- szz-siso 3000 Hz
- h (syy-sxx)/d 0.0
43Deuterium NMR to probe ligand orientation
44Oriented samples ligand orientations
B0
B0
45A 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
46Conformation 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
47Orientation 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
48PISEMA 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
49PISEMA spectra of Fd coat protein
50Tilt 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
51Assignment of PISEMA spectra
PISEMA Spectra of amino acid selectively labelled
Fd cost protein (Marrassi, 2002)
52Extracting 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
53Dipolar waves Fd coat protein
- Breaks in wave indicate
- Start of new secondary structure
- Deformation in secondary structure (kinks in
helices)
54Summary
- 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
55References
- 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.
56Acknowledgements
- 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