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.

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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