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Exploiting spectral anisotropy in membrane studies
Dr Philip WilliamsonMay 2009
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
2
Introduction to anisotropic interaction
3
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
4
Which interactions in NMR
5QDipolarCSAJCS HHHHHH
Isotropic Anisotropic
JCS HHH
DipolarCSAJCS HHHHH
CSAJCS HHHH
CSHH
Chemical Shielding Anisotropy
• Perturbation of the magnetic field due to interaction with surrounding electrons
• Inherently asymmetric (e.g. electron distribution surrounding carbonyl group)
6
Describing interactions: tensors
• Second rank tensors
7
zz
yy
xx
PAS
00
00
00
zzzyzx
yzyyyx
xzxyxx
i j k
i
j
k
x
y
z
zz
yy
xx
Chemical Shielding Anisotropy
• We can describe the perturbation of the main field (B0), by the second rank tensor,
• The Hamiltonian which describes the interaction with the modified field is:
Which can be written in a simplified form as:
8
0
0
0ˆ,ˆ,ˆ
B
IIIH
zzzyzx
yzyyyx
xzxyxx
kkzkykxkCSA
0ˆ BIH k
kkkCSA
0
0
0
B
B
zzzyzx
yzyyyx
xzxyxx
S
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 zz in the laboratory frame.
However, is usually defined in the principle axis system (PAS) not in the lab frame (LF). Therefore, we need to transform from the PAS to LF.
9
0)(ˆ BIH k
zzk
kzkCSA
0)(
12 kzz
Transformation matrix
Can derive a rotation matrix which bring about the rotation described above:
To determine in the laboratory frame, need to apply to the chemical shielding tensor in the principle axis system:
This can be simplified to give general Hamiltonian for CSA in lab frame of:
10
cossinsinsincos
sinsincoscossincossinsincoscoscossin
cossinsincoscoscossinsinsincoscoscos
,,R
),,(),,( 1 RR PASLAB
kzisokCS IBH ˆ2cossin1cos32
220
Effect on resonance position
11
z
x
y
zz =3000Hz
yy=-1500Hzxx =-1500Hz
iso = 1/3(xx+yy+zz) = 0Hz
= zz-iso = 3000 Hz
= (yy-xx)/
kzisokCS IBH ˆ2cossin1cos32
220
/2
Powder Patterns
• In powders we have a random distribution of molecular orientations.
• Thus the lineshape is the weighted superposition of all the different orientations:
12
..sin),,,(8
1)(
2
0 0
2
02 tsts
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.
13
B0
1
2
)cos31(1
42
21312
0
r
E
21
122121212
21312
210
ˆˆ
.ˆ.ˆ3ˆˆ4
IDI
rIrIr
IIr
HD
200
010
001
4 312
210
rD
Orientation dependence of dipolar interactionHomo-nuclear Dipolar Hamiltonian:
Hetero-nuclear Dipolar Hamiltonian:
14
2121
2
312
210,
ˆˆˆˆ32
)cos31(
4IIII
rH zzIID
zzISD II
rH 21
2
312
210,
ˆˆ22
)cos31(
4
dip=20 kHz
dipdip
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:
1.A good reporter on the local electronic distribution about the nucleus (e.g. H-bonding status)
2.Due to large anisotropy, good reporter for orientation studies
15
kkQ IQIHˆˆ
zz
yy
xx
V
V
V
II
eQQ
00
00
00
)12(2
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:
16
)1(ˆ32cossin1cos32
222 IIIH ZQQ
Q
Orientation dependence of a single crystal of Ala-d3
Powder spectrum of Ala-d3
Q
Powder samples
17
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
18
Scaling of anisotropic interactions• Can use different motional models to study averaging of anisotropic
interactions:
– Multisite jump
– Rotational diffusion ....
19
)0,,( PMPMPM )0,,( MDMDMD
Peptide long axis
Membrane normal
2 23cos 1 cos 2 sin2
2H-NMR dynamic studies of acetylcholine salts
BrAChBr AChCl AChClO4
• Temperature dependent
• Lineshapes dominated by motions about the C3 and C3’axis of rotation
• Lineshape provide information about energy barriers associated with rotation
Field (B0)
C3
C3’
Dynamics of 2H-BrACh whilst resident in the binding site on the nAChR
Rotation of quaternary ammonium group hindered in the binding site
Membrane reorientationBackbone dynamicsC3/C3’ Rotation
Reduction in backbone dynamicsC3 or C3’ rotation hindered
C3 and C3’ rotation hindered
ACh Perchlorate Bound BrACh
Cys192/193
Field (B0)
C3
C3’
Structure of the TMD of the nAChR
M4 M3
M1M2
(Ortells, 1999)
Ala8-D3
Leu11-C1
Gly15-N
Gly23-C2
Averaging of anisotropic interactions in DoMPC vesicles
L phase
L phase
15N-Gly1513C1-Leu11
2H3-Ala8
MASMAS Static
Static MAS Static
Structure from dynamics in non-oriented systems
)0,,( PMPMPM
)0,,( MDMDMD
Peptide long axis
Membrane normal
13C1-Leu11
15N-Gly15
Secondary Structure of the M4-TMD
190 200 210 220 230 2406
4
2
0
2
4
6
8
10
12
MR
E (
md
eg
cm
2 dm
ol -
1 )
Wavelength (nm)
CD Spectroscopy indicates
• Over 50% of residues in a -helical conformation
• Conformation preserved in TFE and lipid bicelles
Membrane protein dynamics: APP
Changes in lipid composition:1)Lipid metabolism (Chol/Sph)2)Lipid oxidation3)Level of saturation
amyloid
amyloid
Protease cleavage site accessibility
3.60nm
2.30-2.90nm
Lipid induced elevated -amyloid levels
Increase in bilayerthickness
Change in oligomeric state
-amyloidProtection from -secretase
Orienting Biological Membranes
29
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
n
Mechanical orientation of synthetic lipid bilayersLipid/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)
Salt solutions for maintaining hydrationSaturated aqueous solution with
considerable precipitates% relative air humidityabove 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
Mechanical orientationOriented 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
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
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
B0
Below phase transition, mixed micellar
)1cos3(....)( 2212
031 BNF
n
Above phase transition, discoidal particles - bicelles
Macroscopic orientation of the M4-TMD in DoMPC:DoHPC bicelles
-30 -20 -10010203031P Chemical Shift (ppm)
-30-20-10010203031P Chemical Shift (ppm)
+M4
Do
MP
C
Do
HP
C
Do
MP
C
Do
HP
C
• Positive diamagnetic anisotropy of protein does not perturb alignment• Lineshape analysis indicates a mosaic spread of <4º (limited by intrinsic linewidth)
5 10 15 20 25 30 35 40-12
-10
-8
-6
-4
-2
0
Temperature(ºC)
31P
Ch
em
ica
l Sh
ift (
ppm
)
DoHPC
DoMPC+M4-M4
Flipping the bicelle: advantages for NMR
Conventional BicellesBilayer normal perpendicular to field
•Anisotropy halved (S=-0.5)
•No rotation leads to cylindrical distribution
Parallel bicellesBilayer normal parallel to
field
• Full anisotropy (S=1.0)
• Uniaxial distribution
B0
B0
Flipping the bicelle
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
DMPC
DHPC
DMPC
DHPC
DMPE-DTPA
Prosser, 1998
DMPC/Tm3+=150
DMPC/Tm3+=40
DMPE-DTPA/Tm3+=1
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)
Macroscopic orientation of native membranes
B0
n
n
Native nAChR membrane, pelleted onto Mellanex sheet, 25000 rpm overnight, no drying (Analytical Biochemistry, 1998)
Applications of oriented samples
41
Effect on resonance position
42
z
x
y
zz =3000Hz
yy=-1500Hzxx =-1500Hz
iso = 1/3(xx+yy+zz) = 0Hz
= zz-iso = 3000 Hz
= (yy-xx)/
kzisokCS IBH ˆ2cossin1cos32
220
/2
Deuterium NMR to probe ligand orientation
43
Cys192/193
Field (B0)
C3
C3’
Orientation
0°
90°
Oriented samples – ligand orientations
B0 B0
Orientation±5° Mosaic Spread±5° Orientation±5° Mosaic Spread±5°
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
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
46
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
47
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
1H
X
/2)X
-Y Decouple
35.5º-X
Y+LG -Y-LG
X X -X
m
PISEMA spectra of Fd coat protein
/2)X35.5º
-X
1H
X
-Y DecoupleY+LG -Y-LG
X X -X
m
Tilt of helices from PISA wheels
PISA
Polarity Index Slant Angle
Position of wheels in PISEMA spectra give
orientation of helices in samples
50
TMD 30º with respect to
bilayer
Amphipathic helix on bilayer surface
Assignment of PISEMA spectra
PISEMA Spectra of amino acid selectively labelled Fd cost protein (Marrassi, 2002)
Extracting structure: dipolar waves
Dipolar waves
• dipolar coupling verses residue
• periodicity arises from repeating structure (e.g. -helix)
•enables comparisons to be made with rdc’s in solution
•disruption in ideal nature of secondary structure readily apparent
52
Dipolar waves: Fd coat protein
Breaks in wave indicate:
•Start of new secondary structure
•Deformation in secondary structure (kinks in helices)
53
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
54
ReferencesAnisotropic interactions1.Principles of NMR in one and two dimensions, Ernst, Bodenhausen & Wokenau
Averaging of anisotropic interaction1.Principles of Magnetic Resonance, C.P. Schlicter
Orienting of biological membranes1.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 A;24A(1):17-37.2.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 Journal;35(3):268-275.3.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 Biochemistry;254(1):132-138. (and references therein)4.Prosser RS, Hwang JS, Vold RR. 1998 Magnetically aligned phospholipid bilayers with positive ordering: A new model membrane system. Biophysical Journal;74(5):2405-2418.
NMR studies of oriented biological membranes1.Ramamoorthy A, Wu CH, Opella SJ. 1999 Experimental aspects of multidimensional solid-state NMR correlation spectroscopy. Journal of Magnetic Resonance;140(1):131-140.2.Marassi FM, Opella SJ. 2003 Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints. Protein Science;12(3):403-411. 3.Kim S, Cross TA. 2004 2D solid state NMR spectral simulation of 3(10), alpha, and pi-helices. Journal of Magnetic Resonance;168(2):187-193. 4.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 Society;125:8928-8935.5.Mesleh MF, Opella SJ. 2003 Dipolar Waves as NMR maps of helices in proteins. Journal of Magnetic Resonance;163(2):288-299.
55
AcknowledgementsUniversity of Southampton
School of Biological SciencesDr Phedra Marius
Garrick TaylorPhillippa Hunnisett
Sarah StephensMaiwenn Beaugrand
Dr Jörn Werner Werner group
Zara LuedkeDr Vincent O’Connor
Prof. Lindy Holden DyeProf. Robert Walker
School of Chemistry
Prof. Malcolm LevittLevitt group
Neil Wells
Funding
University of Southampton
School of Engineering and Computing Science
Dr Maurits dePlanque
University College LondonProf. Steve Wood
ETH, ZurichProf. Beat Meier
Dr Aswin VerhoevenDr Giorgia Zandomeneghi
Meier Group
Dr Stefanie KrämerDr Marco Marenchino
University of OxfordProf. Tony Watts
Harvard Medical School/MGH, BostonProf. Keith Miller