Investigations of Membrane Polypeptides by Solid-state NMR

Spectroscopy: Structure, Dynamics, Aggregation and Topology of Supramolecular Complexes

Burkhard Bechinger

Université Louis Pasteur,CNRS - UMR

Chimie-physique moléculaire et spectroscopie

Strasbourg, France

NMR to study membrane proteins

Solution NMR

Requires fast and isotropic motional

averaging< 120 kDa (TROSY)

NMR to study membrane proteins

Solution NMR

Requires fast and isotropic motional

averaging< 120 kDa (TROSY)

Extended liquid crystalline bilayers

are too big

Solid-state NMR

frozen, dry liquid crystalline

membranes

no physical size limitation

Structure, orientationand dynamics

Solid-state NMR provides information on …

• chemical environment• distances• dihedral angles• orientations in space

Structure, dynamics and topology

Oriented membranesBo

Chemical synthesis of

peptides allows labelling at single sites

Deblock

Activate aa

Couple

resin

resin

resin

resin

to reactor

Wash

Wash

Oriented Samples:Structure and Topology

15N chemical shift alignment of the peptide

bond

Solution and solid-state NMR on the same scale

The 2H quadrupolar splitting2H3-alanine

C

C2H

2H

2H

Q ~ 3 cos2-1

Bo

0°

30°

53°

90°

60°

Similar principles apply for many NMR interactions

Detailed helix alignment from combined 15N and 2H measurements

25 50 75 100 125 150 175

25

50

75

100

125

150

175

rotational pitch angle

tilt

an

gle

ppm 20 0 kHz 10 -10

2 angles

2 measurables

Unique solution from Energy Minimization

+++ + +

+

Tilt 95o, pitch 173o

hydrophobic

hydrophilic

KL14 Model Peptide in Oriented Phosphatidylcholine Bilayers

Lipid 2H (kHz) 15N (ppm)

POPC 6.0 74

DMPC 7.6 73

PC20:1 8.3 73

DOPC 10.8 74

Difference 2o

Dynamics:Rotational Diffusion and

Aggregation

Liquid crystalline membranes

33

22

11

| |

Motion around the membrane normal

Rotational averaging:Effect on 15N powder pattern line shape

p p m501 5 0 1 002 5 0 20 0

3 3

1 1

2 2

| |

2 2

33

2211

Static

Rotation around 33 (helix long axis)

Rotation around 22

3322

11

Powder pattern provide orientational information

2H solid-state NMR2H3-alanyl

Bo

Bo

Freezing Rotational Diffusion

50 0 kHz

313 K

303 K

293 K

283 K

263 K

TM helix

IP helix

Loss of intensity during transition

Equilibrium: Mono- / oligomer

20 0 kHz 10 -10 -20 20 0 kHz 10 -10 -20

2H-NMR

Bo

Bo

2H NMR of ‘‘real‘‘ samplese.g. viral channel peptides

20 0 kHz -20 20 0 kHz

Influenza M2 Vpu

2H spectral line shape and mosaic spread

Tilt angle: 10o 40o 50o 700

Mosaic Spread

0.5135

1015

Model amphipathic helix

20 0 kHz 10 -10 -20

= 45.3o or 65.5o

Mosaic spread = 1o

Example:Controlling Topology

Oriented 15N solid-state NMR:LAH4 pH-dependent molecular

switch

pH > 7

pH < 5

pH 6.1

Example: Domain of ICP47

• Herpes simplex virus• 87 residues early gene product

(domain 2-34 active)• Inhibits transport by TAP of antigenic peptides to

surface and thus presentation by MHC I lack of immunogenic response• Solution NMR:

Helix (5-14)-loop-helix (22-31) in SDS micelles

c/o Robert Tampé - Frankfurt

15N solid-state NMR of ICP47(2-34) in oriented POPC

Helix1

Loop

Helix2

L5

E6

M7

A8

D9T10

F11

L12

D13

N14

L5

E6

M7

A8

D9T10

F11L12

D13

N14helix 1

Y22

A23

D24

V25

V25

R26

A,N27

E28

I29

N30

K31

Y22

A23

D24

R26

A,N

27

E28

I29

N30K31

helix 2

‚Modelling‘tilt 84o

tilt 75o

2H solid-state NMR of ICP47(2-34) in oriented POPC

Mosaic spread 10-15o

Model for membrane-bound ICP47

Acknowledgements• Christopher Aisenbrey• Christina Sizun• Bas Vogt• Jesus Raya

• Gérard Nullans, ULP-INSERM Neurochimie

• Robert Tampé, Universität Frankfurt

€ ARC, ANRS, Vaincre la MucoviscidoseRegion Alsace

CNRS, Ministère, ACI Jeune Equipe

Methods to orient lipid bilayers

NewGlaubitz et al.

Combine MAS and

oriented samples

MAOSS at 10 kHz 31P NMR of oriented bilayers

10 kHz

565 Hz

simulated

MAS side band analysis provides

orientational information

Powder

Paralle l

Perpendicular

Experim ent

MAOSS of hydrophobic model peptide in phospholipid bilayer

2 5 0 2 0 0 1 5 0 1 0 0 5 0 p p m

P D P D P D

3.7o mosaic

20 % powder

2 5 0 2 0 0 1 5 0 1 0 0 5 0 p p m

15N NMR 31P NMR

=10o

=25o

P D

P D P D

p p m2 03 0 1 0 0 -2 0

Summary

• MAOSS with new sample set up

low or fast spinning

• spinning side band analysis

tilt, mosaic spread and powder pattern contributions

Model for membrane-bound ICP47

14

22

5

31