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![Page 1: Investigations of Membrane Polypeptides by Solid-state NMR Spectroscopy: Structure, Dynamics, Aggregation and Topology of Supramolecular Complexes Burkhard.](https://reader036.fdocuments.us/reader036/viewer/2022062517/56649f1b5503460f94c31714/html5/thumbnails/1.jpg)
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
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NMR to study membrane proteins
Solution NMR
Requires fast and isotropic motional
averaging< 120 kDa (TROSY)
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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
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Solid-state NMR provides information on …
• chemical environment• distances• dihedral angles• orientations in space
Structure, dynamics and topology
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Oriented membranesBo
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Chemical synthesis of
peptides allows labelling at single sites
Deblock
Activate aa
Couple
resin
resin
resin
resin
to reactor
Wash
Wash
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Oriented Samples:Structure and Topology
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15N chemical shift alignment of the peptide
bond
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Solution and solid-state NMR on the same scale
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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
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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
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Unique solution from Energy Minimization
+++ + +
+
Tilt 95o, pitch 173o
hydrophobic
hydrophilic
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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
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Dynamics:Rotational Diffusion and
Aggregation
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Liquid crystalline membranes
33
22
11
| |
Motion around the membrane normal
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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
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2H solid-state NMR2H3-alanyl
Bo
Bo
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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
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Equilibrium: Mono- / oligomer
20 0 kHz 10 -10 -20 20 0 kHz 10 -10 -20
2H-NMR
Bo
Bo
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2H NMR of ‘‘real‘‘ samplese.g. viral channel peptides
20 0 kHz -20 20 0 kHz
Influenza M2 Vpu
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2H spectral line shape and mosaic spread
Tilt angle: 10o 40o 50o 700
Mosaic Spread
0.5135
1015
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Model amphipathic helix
20 0 kHz 10 -10 -20
= 45.3o or 65.5o
Mosaic spread = 1o
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Example:Controlling Topology
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Oriented 15N solid-state NMR:LAH4 pH-dependent molecular
switch
pH > 7
pH < 5
pH 6.1
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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
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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
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2H solid-state NMR of ICP47(2-34) in oriented POPC
Mosaic spread 10-15o
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Model for membrane-bound ICP47
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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
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Methods to orient lipid bilayers
NewGlaubitz et al.
Combine MAS and
oriented samples
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MAOSS at 10 kHz 31P NMR of oriented bilayers
10 kHz
565 Hz
simulated
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MAS side band analysis provides
orientational information
Powder
Paralle l
Perpendicular
Experim ent
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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
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Summary
• MAOSS with new sample set up
low or fast spinning
• spinning side band analysis
tilt, mosaic spread and powder pattern contributions
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Model for membrane-bound ICP47
14
22
5
31