- EPJ Web of Conferences

EPJ Web of Conferences 30, 03004 (2012)
DOI: 10.1051/epjconf/20123003004
© Owned by the authors, published by EDP Sciences, 2012
džƉĞƌŝŵĞŶƚ ĂŶĚDŽĚĞůůŝŶŐ ŝŶ^ƚƌƵĐƚƵƌĂůEDZ
EŽǀĞŵďĞƌ ϮϴƚŚʹ ĞĐĞŵďĞƌ ϮŶĚ ϮϬϭϭ
/E^dEʹ ^ĂĐůĂLJ͕&ƌĂŶĐĞ
Hartmut Oschkinat
Leibniz Institut
für Molekulare Pharmakoligie
Germany
Biosolid state NMR
[03004]
Organized by
Thibault Charpentier
Patrick Berthault
Constantin Meis
[email protected]
[email protected]
[email protected]
Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20123003004
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Bio-SolidState
Saclay, 30th of November 2011
Hartmut Oschkinat
Leibniz-Institut für Molekulare Pharmakologie
Berlin
Solid-state NMR as a tool for ‘difficult’
areas of structural biology
Heterogeneous, filamentous systems:
Æ cytoskelet-associated proteins
Æ protofibrils
‘Dynamical complexes’
Æ f-actin and associated proteins
Æ focal adhesions
Æ full-length aB-crystallin
Membrane protein complexes in native lipid
environments
Æ all states of one ABC-transporter
ATP ADP+Pi ATP ADP+Pi
03004-p.2
NMR on membrane proteins
Bilayers and
liposomes:
Solid-state
NMR
Micelles:
Solution NMR
Principles of solid state NMR
to study biological membranes
Spin Hamiltonian given as:
Htotal= HZ + HCS+ HJ+ HD+ HQ
Zeeman interaction (HZ)
chemical shielding (HCS),
J-coupling (HJ)
dipolar coupling (HD)
quadrupolar coupling (HQ)
In solids: anisotropic contributions (<3cos2q-1> dependence); q is angle
between the angle of the tensor element with respect to the applied field, Bo.
For large (Mr » 30k) and slowly tumbling molecules (t r > ~10's kHz), with all
orientations of the nucleus present at any one time, give rise to broad, powderlike NMR spectra which are an envelope of the individual orientational
frequency dependent lines.
Anisotropy is often exploited in a biomembrane system
03004-p.3
Experimental Observation
B0
Orientation Studies: 15N labelling
The Hamilton Operator of the Dipolar Interaction
and the Dipolar Alphabet
The Hamilton Operator of the dipolar interaction
for 2 particles with spin 1/2 is given by
^ DD
H IS
­ 3 & &
&&½
= bIS® 2 I rIS SrIS − I S¾
¯ rIS
¿
( )( )
with
bIS = − μ 0
γ Iγ S !
4 πrIS3
γ ≡ gyromagnetic ratio of the nuclei
! ≡ Planck䇻s constant
^ DD
γ γ ! 3cos2 β − 1
(3I z Sz − I 㽢S)
H IS = μ 0 I S 3
μ0 ≡ magnetic permeability
4 πrIS
2
rIS ≡ distance between I and S.
03004-p.4
Effects of magic angle spinning on solids
54.7㼻
magnetic
field
MAS
Speed, ωr
1
1
1
54.7㼻
NMR Probes for solid state NMR
7mm i.d. sample rotor; 400MHz HXY probe
03004-p.5
Practicalities of magic angle sample spinning
Sample contained in rotor 1.2 - 9mm diameter;
Usually fully hydrated; ~ 10 - 300nmoles of sample of interest; Usually
labeled with 13C, 15N, 2H, 19F, etc
Physics of Dipolar Recoupling
M. H. Levitt et al. J. Chem. Phys. 92, 6347 (1990).
X. Feng et al. Chem. Phys. Lett. 257, 314 (1996).
03004-p.6
Typical magnitude in ppm of magnetic
interactions for various nuclei
Magic angle spinning solid state NMR
narrows anisotropically broadened spectra
13C
NMR
magic angle 54.7㼻
Static NMR spectrum
MAS NMR spectrum
Anisotropy broadened
(in chemical shift/
dipolar couplings)
Anisotropy averaged
by spinning
(wr ~ 1 - 15kHz)
Small (Mr ~ 3k) peptide in fluid lipid bilayers
03004-p.7
90
13C
Membrane protein structures by solidstate NMR
Samples
and
Strategy
03004-p.8
Systems with short-range order can be
investigated by solid-state NMR
3roteins in a
micro-crystalline state
e.g. SH3 domain
2D crystals of
membrane proteins
e.g. OmpG
Ligand /cofactor bound
to a receptor
e.g. NT II + nAchR
Amyloid fibrils
Reconstitution into lipid bilayers and
two dimensional crystallisation by dialysis
+
E. coli total lipid extract
detergent
mixed lipid detergent micelles
refolded protein
2D crystals
03004-p.9
LPR
1.0
3k
- dialysis 6 days
‘298 K’
400MHz
1.0
45 k
- dialysis 8weeks
LPR
0.75
45 k
- dialysis 8weeks
‘298 K’
400MHz
0.5
45 k
- dialysis 8weeks
03004-p.10
900 MHz
α-spectrin SH3 Domain
Barely soluble at pH 7
Precipitates in microcrystalline
form by changing from 3.5 to 7
03004-p.11
Protein structures by solid-state NMR
ASSIGNMENTS
Proton-driven spin diffusion (PDSD) experiment
to detect 13C-13C and 15N-15N long-range
correlations
1
H
TPPM
CP
tmix : from 15 to 500
ms
CP
t1
t mix
t2
03004-p.12
Identification of the single amino acids
γ
α
80
60
40
20
0
0
threonine
γ
β α
HO
NH2
20
γ
O
V58Cγ2-Cβ
V58Cγ1-Cβ
V58Cγ2-Cα
V58Cγ1-Cα
T24Cγ-Cβ
V58Cγ2-Cγ1
20
T24Cγ-Cα
40
OH
α
β
80
60
α
30
60
40
0
80
V58Cβ-Cα
40
γ1,γ2
13
β
20
C (ppm)
β
50
80
60
valine
γ1
γ2
β α
NH2
40
β
20
0
0
γ1,γ2 20
O
T24Cβ-Cα
60
40
OH
α
80
70
60
60
40
20
0
80
70
60
50
40
13
30
20
C (ppm)
Chemical shift assignment
• Identification of
the single amino acids
3
3
‡ Sequential assignment
‡ Proton assignment
Based on the 13C and 15N resonance assignment
03004-p.13
Chemical shift assignment
• Identification of
the single amino acids
3
3
‡ Sequential assignment
‡Æ
Protonless MAS NMR requires thus a better
definition of the amino acid type prior to the
sequential assignment
Resonance assignments using u-13C,
15N-labelled protein
13C, 15N
Backbone assignments:
NCA
NCO
13C-PDSD
13C
NCACX (specific CP!)
NCOCX
15N-PDSD
Side chain assignments:
13C-PDSD
1H (13C)
13C-RFDR
1H (15N)
assignments:
1H-15N
assignments:
FSLG/PMLG
3D 1H-15N-13C HNCA
1H-13C
FSLG/PMLG
1
13
3D H- C-13C FSLG-RFDR
03004-p.14
NCO and NCA spectra of (U-13C,15N) α-spectrin SH3
domains
17.6 T, 278 K,
12 kHz MAS
[ppm]
G51/Q50
V53/F52
V9/L8
R21/P20
A56/A55
Y57/A56
D40/K39
G28/K27
Y15/D14
T32/L31
E22/R21
I30/D29
L8/E7
R49/D48 K27/K26
W41/D40
K18/E17
S36/N35
L61/K60
N38/T37
K60/K59
L12/A11
Y13/L12
V53
V58
V58/Y57
S19/K18
V23/E22
T37/S36
T37
T24/V23
Q50/R49
F52/G51
K59/V58, E45/V44
K43/W42
M25/T24, K39/N38
D29/G28
E17/Q16
V44/K43
L10/V9
K26/M25 W42/W41
L34/L33
Q16/Y15
T32
I30
K27
V44
L61
E7,E17 K18
S36
L12
A55
L33/T32
L33
NCO
190
180
170
160
65
13
60
120
L10
W42
K26
L34, N38
Q16
A11
L31
K60
A11/L10
N
115
G28
K59
K43 M25
L8
D29 R49
W41
E22
K39
15
110
R21
A56
S19 Y57
N35
D48
Y15
D40 Q50
F52
D14
E45
T24
D14/Y13
Y13
V9
V23
N35/L34
D48/N47
L31/I30
A55/P54
G51
125
130
NCA
55
50
45 [ppm]
13
C
C
13
PDSD, NCOCX and
NCACX spectra of
(U-13C,15N)
SH3 domains
T32 α
T32 β
T32 CO
T32 γ
A55
T24
T24
T37
T32
T37
V23
V23
V44
V58
T32
A55
V53
V44
A56
A11
[ppm]
V53
A56
A11
V58
V9
V53
V58
V44
V44
V9
V58
V9
V9
15
V53
V44
V23
I30
V58
V23
V23
V53
20
V9
I30
I30
P20
Q50
E45
D40
P54
E45
E7
W41
E17 V53
E45
V23
V44
V58
P20
Q50
E17 Q50
E22
Q16 E45
E7
E17
E22
E22
E17
N35/N38
D14
D14
D29
E45
P20
P54
Q16
D48
N35/N38
D29
Q50/W42
Q16 Q50 P20
V9
E7
E17
E17
E22
K27
K60
K59
K59
M25
E22
N38
Y13
D40
D40
K26
K59
K27/K43
K18/K60
K27
K60
Y57
D48
N35
D14
D29
F52
E45
E7/M25
M25
E22
R21/R49
17.6 T, 278 K
P20
L10
P54
L8
L31
L61
L33/L34
S19
R21/R49
L10 L10 L10 A11
K26/K59
K43
L8
L8
L61
L61
S36
I30
T24
F52
T32
S36
50
A56
A55
55
60
I30
K27/K60 K60 K27
K27
L33
K39/K60
K18 K18/L34
T24
T32
T37
S19
T37
45
L33 L34
L12 L12
L34
L33
L12
I30
K18
L12
35
40
L10
§
L10
#
25
30
I30
I30
K26
K26
K18
K18
R21/R49
I30
K43
K43
L61
L61 L61 L12/L33
K
L12
L12
L33/L34
L33 L34
L34 L8 L8
R21/R49 L8
L10
C
65
T32
T32
T37
T24
T32
1
T37
PDSD
H
T24
y
C
x
S19
T37
X
T32/T32
T32 β
T32α
1
ϕ2
y
ϕ3
ϕ4
190
V23
V23
D40
G28
Q50
F52 E22
D14
K59
E22
K43 M25
I30
L8
E7
D29, R49
R49
W41
L10
N38
W42
K26
K26
L61, N38 L61
K60
Q16
L34
A11
L31
A55
Q50
K59
170 75
13
C
03004-p.15
110
A56
115
T24
K59
T32 γ
E45
I30
M25
K39
K27 L10
K27
V44
W41 R21
L61
L34
K60 L61/L34
K60
Q16
L12
L31
120
I30
V44
125
A11
A55
130
70
65
135
NCACX-PDSD
P20
180
V23
T37
V53
L33
NCOCX
-y
V53 V58
V9 V58
D48
F52
E45
L33/T32
x
V9
R21
A56
x
15
C
S19
I30
E7, E17
K39
K27
V44 K18
S36
S36
L34
K60
L12
ϕ
13
Y13
Y57
N35
Y15
T24
T32/L31
V53
V58
V9
V23
A56/A55
N
15
[ppm]
G51
-y
T37
H
75
x
13
1
70
PDSD
T24
T37
60
55
50
45
40
35
30
25
20
15
[ppm]
13
C
N
NCOCX and NCACX spectra of (U-13C,15N) SH3 domain
15
G51/Q50
G51
V53/F52
N
[ppm]
G51
V53
V53
110
T37 β
115
Q50
Q50
F52
F52
K39 β
F52/G51
120
N38 β
125
17.6 T, 278 K
8 kHz MAS
130
NCOCX
P54
NCACX-PDSD
P20
180
1H
15 N
15
170
13
C
TPPM
V58
V9
V23
x
t1
-y
I30
V44
D40
E45
15 N
x
TPPM
V58
D48
D40
G28
Q50
D14
Y15
Y57
110
V23
115
Q50
D14 E22
F52
E45/K59
120
K39
I30
K43
K43
M25
M25
M25
K27
D29, R49
D29 V44
W41
R49
W41
K18
L10
L10
L10 L34
E17 W42
W42
K26
K26
L34
L61,
N38
L61, N38
K60
Q16
Q16
L12
A11
L31
L31
L31
E22
L33
A55
L33
x
N35
K59
K39
V53
V9
A56
D48
K27 E7,E17
S36
S36
L34
K60
L12
1H
Y13
Y13
S19
Y57
N35
F52
T32
t2
V53
Y15
T24
y
13C
T37
TPPM
CW
N
[ppm]
G51
S19
x
125
130
L33
TPPM
CW
135
13C
75
70
65
135
NCACX-BD
P20
t1
60
55
50
45
40
35
30
25
20
15 [ppm]
13
1H-13C-correlations
by the frequency-switched
Lee-Goldberg technique
π /2
θm
2π
1
H
+ ΔLG −ΔLG
+X
13
2π
+X
-X
C
LG-CP
+Y
+Y
RAMPCP
t2
z
A ~
z
B
θm
x
z
x
x~
y, ~
y
y
03004-p.16
2D 1H-13C FSLG of 13C-15N SH3 domain
(11.5 kHz MAS)
Tcp=350 μs
Tcp=2.0 ms
Assignment of amide protons from 3D HNCA
spectrum
400 MHz (9.4 T)
MAS : 8 kHz
T = 280 K
03004-p.17
Solid-state NMR of full-length αB-crystallin
α-crystallins (αA-crystallin and αB-crystallin)
where discovered in 1896 in the eye lens
They are together a chaperone system for
β- and γ-crystallin.
cataract
αB-crystallin is distributed ubiquitously, acts as a
sHSP and mutants are involved in many diseases.
(e.g., multiple sclerosis, Alzheimers disease, cardiomyopathie)
aB crystallin forms dynamic, polydisperse oligomers
of 24 – 32 subunits, ~600kDa, with dimers as basic building blocks
alpha-B crystallin is a small heat shock protein
S-W-F
N-terminus
I-X-I
conserved core domain
(α
α-crystallin domain, ~100 aa)
extension
tail
176 amino acids
EM Structure at 3.6 nm
resolution
adapted from Haley et. al..
J. Mol. Biol. (1998)
03004-p.18
Dataset/approach
3 Samples:
uniformly 13C,15N
made from 1,3 - 13C - glycerol
made from
2 - 13C - glycerol
With help from Ponni Rajagopal,
Klevit Lab in Seattle!
- 3D NCACX (NiCi), NCOCX (NiCi-1)
- 3D NCACB (NiCi), NCACBCX (NiCi)
- 2D 13C-13C correlations (PDSD)
- J-decoupling
- Methyl-Filtering
L137N-Cα-Cγ
L137N-Cα-Cδ∗
15
1
L137N-S136C-Cβ
δ N(L137)=122.9 ppm
1,3G - NCOCX
b)
τmix = 75 ms 171
2
L137N-S136C-L137Cα
L137N-S136C-Cβ
δ15N(L137)=122.9 ppm
U - NCOCX
c)
τmix = 35 ms 171
K103N-G102C-Cα
L137N-S136C-Cα
15
δ N(S136)=118.3 ppm
U - NCACB
d)
τmix = 2 ms
57
S136N-Cα-Cβ
15
S136N-S135C-Cβ
δ N(S136)=118.3 ppm
U - NCOCX
e)
τmix = 35 ms 173
S76N-F75C-Cβ
S136N-S135C-Cα
15
S135N-Cα-Cβ
E87N-Cα-Cβ
15
δ N(S135)=112.1 ppm
2G - NCACX
τmix = 200 ms 57
g)
S135N-Cα-T134C
3
δ N(S135)=112.1 ppm
U - NCACB
f)
τmix = 2 ms 57
S135N-Cα-T134Cα
4
15
S135N-T134C-Cα
S135N-T134C-Cβ
G102N-H101C-Cα
S135N-T134C-Cγ2
δ N(S135)=112.1 ppm
U-NCOCX
τmix = 35 ms
h)
173
δ15N(T134)=117.3 ppm
T134N-Cα-C
T134N-Cα-Cβ
T134N-Cα-Cγ2
S139N-Cα-Cβ
03004-p.19
U-NCACX
τmix = 25 ms
61
i)
13
δ N(L137)=122.9 ppm
U - NCACX
τmix = 25 ms
a)
53
C(ppm)
15
L137N-Cα-Cβ
Structure of alpha-B crystallin in functional oligomers
Intermolecular N- and C-terminal
interactions are responsible for
oligomerisation
SAXS investigations
An oligomer model can be reconstructed, assuming tetrahedral
symmetry
SAXS
Space filling
of dimer
03004-p.20
N-terminus
conserved core domain
(α-crystallin domain, ~100 aa)
extension
tail
Monomer X
S59A
F118A
T134K-T144R
N78G-P86A
P TSSLSSADGVLTVN
VNLDVKHFSMonomer
ΔP155-E165
PERTIPITREE
T
I
P
I
T
pH modulation of the chaperone binding site
pH 7.5
pH 6.5
03004-p.21
Solution NMR of flexible residues
SAXS measurements at different pH
03004-p.22
Conclusions
The structure suggests that the activity of αB-crystallin
is tightly connected to its oligomeric state. An intact
oligomer should not be active.
structural changes at pH 6.5, 9.0 and 4.5 suggest
regulation by pH
Protein structures determined by MAS solid-state NMR
1M8M, 62 aa
SH3 domain
Oschkinat
1RVS, 10 aa
Transthyretin
Jaroniec et al. 2004
1XSW, 38 aa
Kaliotoxin
Lange et al. 2005
2E8D, 4x 22 aa
beta2-microglobulin
Iwata et al. 2006
2JSV, 56 aa
GB1 (VEAN)
Franks et al. 2007
2JU6, 56 aa
GB1 (3D protons)
Zhou et al. 2006
2JZZ, 76 aa
Ubiquitin
Manolikas et al. 2008
2K0P, 56 aa
GB1 (CS)
Robustelli et al. 2008
2K9C, 152 aa
MMP-12 (PCS)
Balayssac et al. 2008
2KHT, 30 aa
alpha defensin HNP-1
Li et al. 2010
2KIB, 8x 7 aa
hIAPP
Nielsen et al. 2009
2KRJ, 152 aa
MMP-12
Bertini et al. 2010
2NNT, 4x 31 aa
ww2 domain
Ferguson et al. 2006
2RLZ, 2x 85 aa
Crh dimer
Loquet et al. 2007
2RNM, 5x 71 aa
HET-s prion
Wasmer et al. 2008
2UVS, 38 aa
Kaliotoxin
Korukottu et al. 2008
2KLR, 2x 82 aa
aB crystallin
Jehle et al. 2010
2KWD
GB1 (TEDOR
Intermolecular)
Nieuwkoop et a. 2010
2KQ4, 56 aa
GB1 (3D TEDOR)
Nieuwkoop et al. 2009
2KJ3, 3x 71 aa
HET-s prion
Van Melckebeke et a.
2010
03004-p.23
Quality of MAS solid-state NMR structures
Whatif quality Z-scores
aB
crystallin
HETs
hiAPP
HETs
K3 β2
WW2
GB1
TTR
2
MMP1
GB1
HNP-1
2
MMP1
GB1
Ubq
Crh
GB1
GB1
KTX
KTX
SH3
Solid-state NMR vs. X-ray vs. solution NMR
2K0P = GB1 from ssNMR chemical shifts only
2KLR = aB crystallin dimer
2KRJ = MMP-12 (CHHC + PDSD/DARR + PAR +PAIN + PCS)
Molprobity score= f(clashes, rotamer outliers, Ramachandran non-favored)
46
03004-p.24
NMR on membrane proteins
ArtMP, an ABC transporter
Arginine import system of Geobacillus stearothermophilus
ATP ADP+Pi
ATP ADP+Pi
The holy grail:
All functional states of one
system in native lipid bilayers
03004-p.25
Preparation of ArtMP
Expression in E.coli
Lipid extraction of
G. stearothermophilus
Reconstitution
2D Crystallisation
ATP ADP+Pi
ATP ADP+Pi
Magic-Angle Spinning (M
dr
bearing
NMR-Spectra of ArtMP-2D-Crystals
$UW033<
ZLWKRXW $73
&&3'6'0+].0LVFK]HLWPV
&SSP
&SSP
9LYLHQ/DQJH
(UJHEQLVVH
03004-p.26
&SSP
&SSP
$UW037<3
ZLWK $73
1H
detection of perdeuterated samples, recrystallized from
H2O/D2O = 1:9: SH3 with 1H, 2H, 13C and 15N
By Rasmus Linser, Vipin Agarwal, Veniamin Chevelkov,
Bernd Reif
Linewidths: 24 㼼5 Hz
Use of standard pulse solution programs
R. Linser et al., Sensitivity Enhancement using paramagnetic
relaxation in MAS solid state NMR of perdeuterated proteins,
J. Magn. Res, subm.
Reconstitution of ArtMP
CP
15N
ATP ADP+Pi
ArtM(P)15N
15N
ATP ADP+Pi
INEPT
03004-p.27
CP
15N
15N
15N
15N
ATP ADP+Pi
Art(MP)15N
ATP ADP+Pi
INEPT
1H
detection of perdeuterated samples, recrystallized from
H2O/D2O = 9:1
By Rasmus Linser, Vipin Agarwal, Veniamin Chevelkov,
Bernd Reif
Linewidths: 24 㼼5 Hz
Use of standard pulse solution programs
R. Linser et al., Sensitivity Enhancement using paramagnetic
relaxation in MAS solid state NMR of perdeuterated proteins,
J. Magn. Res, subm.
03004-p.28
Optimum Levels of Exchangeable Protons in
Perdeuterated Proteins for Proton Detection
in MAS Solid-State NMR Spectroscopy:
A study at 24 kHz spinning
By the way: there are as many
exchangeable sites in the side chains of
the 20 aa as in the backbone!
Effect of 1H% on 1H-Linewidths @ 24kHz-MAS
1H
%
1H
03004-p.29
Average*
FWHM (Hz)
15N
Average
FWHM (Hz)
10
19.0 ± 3.3**
11.3 ± 5.4
20
20.2 ± 5.7
12.4 ± 7.2
30
22.5 ± 6.5
15.4 ± 7.5
40
27.5 ± 6.1
16.9 ± 7.3
60
32.5 ± 10.3
25.1 ± 13.5
80
37.5 ± 9.9
32.3 ± 14.3
100
58.3 ± 20.5
41.2 ± 14.4
Another Issue !!
Remaining & Dissappearing Signals
2D 1H-15N MAS NMR: Double-CP, 24 kHz, 275K, 400MHz
Red: 20%
Black: 60%
Red:
20%
Black: 100%
Remaining & Dissappearing Signals
Remaining:
From the mobile parts,
(L61, D62).
Dissappearing: From the more rigid parts, (V44, A55).
03004-p.30
S/N: CP
S/N per unit-time
1H
%
1H
T1
(s)
Average
S/N Ratio (CP)
S/N Ratio (CP)
per unit-time
10
4.28
18.9 ± 4.7
21.1
20
2.58
33.5 ± 11.4
48.4
30
2.13
41.1 ± 16.4
65.3
40
1.67
44.6 ± 22.6
80.1
60
0.85
26.4 ± 15.7
66.5
80
0.76
18.3 ± 13.9
48.7
100
0.68
11.1 ± 6.3
30.9
Relaxation Delay: 7s.
Dynamic Nuclear Polarization
Trent Franks, Sascha Lange, Arne Linden, Barth van
Rossum
03004-p.31
EWƉƌŝŶĐŝƉůĞ
ŶĞĞĚĞĚĂŵŽƵŶƚϱŶŵŽůͬϮϱђů;Ϭ͘ϮŵDͿ
DNP Prinzip 0
03004-p.32
DNP Prinzip 1b
DNP Hardware
Cooling Cabinet
controls sample
temperature ~95K
Gyrotron
produces
250 GHz
microwaves
Standard
400 MHz
NMR magnet
Gyrotron
controller
3 pressurized
exchangers within
one dewar
liquid N2
reservoir
03004-p.33
LJŶĂŵŝĐEƵĐůĞĂƌWŽůĂƌŝƐĂƚŝŽŶʹ ƐŽůŝĚƐƚĂƚĞʹ EDZ
technique
–
nACh-Receptor
dĞĐŚŶŝĐĂůŝƚŝĞƐ
Different sources of line broadening: radicals, freezing of
sample
Which labelling pattern gives the best enhancement?
Which nuclei should be excited first?
03004-p.34
Effect of the biradical concentration
Which nuclei to excite best?
03004-p.35
Polarisation build-up in [s]
• Buildup behavior is
different for diff. Nuclei
•
1H
< 13C << 15N
• Deuteration increases the
IJB significantly
• Dipolar coupling & spin
diffusion is important.
DNP buildup is also a function of spin diffusion
03004-p.36
^LJƐƚĞŵƐĂŶĚƚŚĞŵĞƐ
Systems and themes investigated in Berlin:
Membrane proteins without purification
Æ Neurotoxin bound to nAchR in native
membranes
Æ Mystic expressed in E.coli membranes
The nascent chain in ribosomes
Retinal in Drosophila eyes
http://www.dkimages.com/discover/previews/824/5020072.JPG http://www.sevcikphoto.com/images/naja_naja_2.jpg
The system
13C, 15N
ďůƵĞ͗ďŝŶĚŝŶŐƐŝƚĞƐĚƵĞƚŽ
<ƌĂďďĞŶĞƚĂů͕͘:D͕ϮϬϬϵ
ŚZ͗
ŵƵƐĐĂƌŝŶŝĐ͗ŵĞƚĂďŽƚƌŽƉŝĐ;'ͲWƌŽƚĞŝŶĐŽƵƉůĞĚ͖ďƌĂŝŶ͕ŚĞĂƌƚ͕ǀĞƐƐĞůƐͿ
ŶŝĐŽƚŝŶŝĐ͗ŝŽŶŽƚƌŽƉŝĐ;ůŝŐĂŶĚŐĂƚĞĚŝŽŶͲĐŚĂŶŶĞůƐͿ
ƉĂƌĂƐLJŵƉĂƚŚĞƚŝĐĂƵƚŽŶŽŵŝĐŶĞƌǀŽƵƐƐLJƐƚĞŵ͕ŶĞƵƌŽŵƵƐĐƵůĂƌũƵŶĐƚŝŽŶ
ƐŝŵŝůĂƌ͗'Ͳ͕'ůƵƚĂŵĂƚͲZĞĐĞƉƚŽƌ͖ĨŝǀĞĚŝĨĨĞƌĞŶƚƐƵďƵŶŝƚƐ
03004-p.37
EĞƵƌŽƚŽdžŝŶ//ďŽƵŶĚƚŽŶĐŚZ
room temperature
DNP
ZĞƐŽůǀĞĚƐƉĞĐƚƌĂ
03004-p.38
ZĞƐŽůǀĞĚƐƉĞĐƚƌĂ
ZĞƐŽůǀĞĚƐƉĞĐƚƌĂ
ƌŶĞ>ŝŶĚĞŶͶ ϳϲ
ϭϱ͘Ϭϱ͘ϮϬϭϮ
03004-p.39
Ed//ĂƚŶĐŚZĐŽŵƉĂƌĞĚƚŽƌŽŽŵƚĞŵƉĞƌĂƚƵƌĞ
10 days
10 hours
dŚĞŶĂƐĐĞŶƚĐŚĂŝŶŝŶƚŚĞƚƵŶŶĞůŽĨƚŚĞƌŝďŽƐŽŵĞ
-the tunnel is ~100‡ long
-with a diameter of ~10‡ at a
constriction point
- and ~20‡ at the widest region
03004-p.40
DsbASecM:
MKKIW LALAG LVLAF SASAA
FSTPV WISQA QGIRA GP
Enolase-SecM:
MSKIV KIIGR REIID SRGNP T FSTPV WISQA QGIRA GP
C7
DsbA-SecM
vs.
13C
Enolase-SecM
Ala
Ser
CP 750 us
Mix 1ms
Exp.Time 3.8d
CP 750 us
Mix 1ms
Exp.Time 4d
Thr
13C
Ser - CĮ :
Ser - Cβ
β:
G(B) = 5,5%
G(B) = 6,8%
G(H) = 48,2%
G(H) = 65,1%
G(C) = 46,3%
G(C) = 28,1%
joint-probability:
Ser (CĮ,Cβ
β) :
P(B) = 0,83%
P(H) = 70,13%
P(C) = 29,04%
03004-p.41
Ser - CĮ :
Ser - Cβ
β:
G(B) = 64,8%
G(B) = 54,8%
G(H) = 0,1%
G(H) = 0,14%
G(C) = 35,1%
G(C) = 54,1%
joint-probability:
Ser (CĮ,Cβ
β) :
P(B) = 69,2%
P(H) = 0,0%
P(C) = 30,83%
Ser - CĮ :
Ser - Cβ
β:
G(B) = 14,9%
G(B) = 26,1%
G(H) = 29,7%
G(H) = 21,4%
G(C) = 55,4%
G(C) = 26,5%
joint-probability:
Ser (CĮ,Cβ
β) :
P(B) = 9,8%
P(H) = 16,1%
P(C) = 74,1%
03004-p.42
^ŽŵĞĞdžƉĞƌŝĞŶĐĞƐ͕ĐŽůůĞĐƚĞĚ
Samples
SH3
Ribosomes
Eyes
NT II
Kinesin
Mystic
Proline
sample amount
[nmol/rotor]
2000
16
2
40
50
30
99
sample
appearance
solid
wet pellet
wet pellet
solid
wet pellet
wet pellet
solid
added glycerol
[μl]
15
5
5
6
15
5
15
S/N (carbonyls)
194
33
23
86
73
62
67
enhancement
39
16
17
10
11
30
38.5
3,00
2,30
1,40
1,90
0,60
1,20
1,90
T1 H [s]
ĐŬŶŽǁůĞĚŐĞŵĞŶƚƐ
DNP (NMR)
Arne Linden
Sascha Lange
Ümit Akbey
Trent Franks
Barth- Jan van Rossum
Colleagues at MIT
Bob Griffin
Torsten Maly
Alexander Barnes
Björn Erzelius
Wet Lab
Nils Cremer
Anne Diehl
and all others
Ludwig Krabben
Hartmut Oschkinat
ƌŶĞ>ŝŶĚĞŶͶ ϴϰ
ϭϱ͘Ϭϱ͘ϮϬϭϮ
03004-p.43
Thanks to ...
• Oschkinat et. al.:
–
–
–
–
–
–
Dr. Bart van Rossum
Dr. Trent Franks
Dr. Ümit Akbey
Sasha Lange
Arne Linden
Dr. Anne Diehl
• Reif et. al.:
–
–
–
–
• .
Prof. Bernd Reif
Rasmus Linser
Vipin Agarwal
Venjamin
DNP-Magnet measurements
DNPers (Dashing Nitrogen Pushers)
Protein structure determination by magic-angle spinning
solid-state NMR
NMR/structure calculation
B. van Rossum
OmpG:
Trent W. Franks
M. Hiller
S. Jehle
W. Kühlbrandt (Fra)
Nascent chain:
S. Lange
Ö. Yildiz (Fra)
Bernd Bukau, Heidelberg
A. Linden
K. Vinothkumar
Anna Rutkowska, Heidelberg
V. Lange
NeurotoxinII/nAchR:
Melanie Rosay, Billerica
R. Kühne
L. Krabben
Leo Tometich, Billerica
J. Pauli
C. Weise, FUB
Bob Griffin, MIT
M. Fossi
F. Hucho, FUB
Thorsten Maly, MIT
N. Schröder
P. Schmieder
WW-Fibrils:
aB-crystallin:
Preparation SH3 and aB-c:
A. Fersht, Cam.
Ponni Rajagopal, Seattle
K. Rehbein
N. Ferguson
R. Klevit, Seattle
A. Diehl
T.D. Sharpe
R. Vernon, Seattle
ABC-Transporter:
M. Petrovich
D. Baker, Seattle
M. Schneider, HUB AND Bernd Reif, Rasmus
H. TidowLinser, Vipin Agarwal, Venjamin Chevelkov..
03004-p.44