NMR to study membrane proteins

advertisement
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
Solid-state NMR
Requires fast and isotropic
motional averaging
< 120 kDa (TROSY)
frozen, dry
liquid crystalline membranes
Extended liquid crystalline
bilayers are too big
no physical size limitation
Structure, orientation
and dynamics
Solid-state NMR provides
information on …
•
•
•
•
chemical environment
distances
dihedral angles
orientations in space
Structure, dynamics and topology
Oriented membranes
Bo
Chemical
synthesis of
peptides allows
labelling at single
sites
resin
Deblock
Activate aa
resin
Wash
to
reactor
Couple
resin
Wash
resin
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 splitting
2H
3-alanine
90°
Bo
DnQ ~ 3 cos2Q-1
60°


53°
Q
30°
Similar principles apply for many NMR interactions
0°
Detailed helix alignment from
combined 15N and 2H measurements



2 angles
2 measurables
175
ppm
20

125
tilt angle
150
100
75
50
25
25
50
75
100
125
rotational pitch angle
150

175
10
0
-10
kHz
Unique solution from Energy Minimization
hydrophobic
hydrophilic
+
+ + +
+
Tilt 95o, pitch 173o
+
KL14 Model Peptide in Oriented
Phosphatidylcholine Bilayers
Lipid
POPC
DMPC
PC20:1
DOPC
2H
(kHz)
15N
(ppm)
6.0
7.6
8.3
10.8
74
73
73
74
Difference 2o
Dynamics:
Rotational Diffusion and
Aggregation
Liquid crystalline membranes
Motion around the membrane normal

||

11

33
22
Rotational averaging:
Effect on 15N powder pattern line shape
2 2
1 1
Static
3 3

Rotation around
33 (helix long axis)
Rotation around
22
33
11 22
||
33 22
2 2
250 200 150 100
11
50
ppm
Powder pattern provide orientational information
2H
solid-state NMR
2H -alanyl
3
Bo
Bo
Freezing Rotational Diffusion
IP helix
TM helix
313 K
303 K
293 K
283 K
Loss of intensity
during transition
263 K
50
0
kHz
Equilibrium: Mono- / oligomer
2H-NMR
Bo
Bo
20
10
0
-10
-20
kHz
20
10
0
-10
-20
kHz
2H
NMR of ‘‘real‘‘ samples
e.g. viral channel peptides
20
0
Influenza M2
-20
kHz
20
0
Vpu
kHz
2H
spectral line shape and mosaic spread
10o
Tilt angle:
40o
50o
700
Mosaic
Spread
0.5
1
3
5
10
15
Model amphipathic helix
20
10
0
-10
-20
kHz
Q = 45.3o or 65.5o
Mosaic spread = 1o
Example:
Controlling Topology
Oriented 15N solid-state NMR:
LAH4  pH-dependent molecular switch
pH < 5
pH 6.1
pH > 7
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
6
D13 E
Helix1
T10
E6
9
D
13
N14
helix 1
M7
L5
L12
11
L
F
8
A
tilt 84o
F
A8
11
‚Modelling‘
A
K31
30
23
D24
N
A,N27
Helix2
A,N27
23
A
24
D
tilt 75o
N14
L5 M7
12
Loop
D
T10
D9
helix 2
R26
R26 E28
E28
22
Y
22
Y
I29
V25
K31
N30
V25
I29
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 Mucoviscidose
Region Alsace
CNRS, Ministère, ACI Jeune Equipe
Methods to orient lipid bilayers
G la u b it z e t a l .
Combine MAS and
oriented samples
N ew
MAOSS at 10 kHz
31P
NMR of oriented bilayers
10 kHz
565 Hz
simulated
Powder
Parallel
Perpendicular
Experiment
MAS side band
analysis provides
orientational
information
MAOSS of hydrophobic model
peptide in phospholipid bilayer
15N
PD
PD
31P
NMR
NMR
PD
PD
PD
PD
250 200 150 100
50
ppm
=10o
3.7o mosaic
=25o
20 % powder
250 200 150 100
50
ppm
30
20 10
0
-20 ppm
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
22
14
5
31
Download