NMR of
large proteins
•
•
•
•
TROSY (up to 100 kDa)
CRINEPT (> 100 kDa)
H/D exchange (up to X MDa)
Membrane Proteins
Chemical shift monitoring
•
•
•
•
•
•
•
Acquisition (Ernst)
CW
Chemical shift evolution (Ernst, Jener)
GFT (Szyperski)
Hadamard Spectroscopy (Kupce)
Off-resonance decoupling
Spin state selective off-resonance decoupling
(SITAR)
• Coded Spectroscopy
1
1D 1H NMR
Jr(H,C)
1
Jr(H,C)
Jr(H,C)
cw
IZ
IX
R2
1
1
ω
IX(t) = IX(0) Cos(ω t) Exp[- R2 t]
Multi-dimensional NMR Spectroscopy:
The problem of chemical shift monitoring in
the indirect dimension
H
N
2
Chemical shift evolution
1H
15N
Ix
t2
t1= 1 in0
-->
IzSx cos[ωS in0]
-->
Ix cos[ωI t2] cos[ωS in0]
Chemical shift evolution
1H
15N
Ix
t2
t1= 2 in0
-->
IzSx cos[ωS 2 in0]
-->
Ix cos[ωI t2] cos[ωS 2 in0]
3
Chemical shift evolution
1H
t2
t1= 3 in0
15N
Ix
Ix
-->
-->
IzSx cos[ωS 3 in0]
IzSx cos[ωS 2 in0]
-->
-->
Ix cos[ωI t2] cos[ωS 3 in0]
Ix cos[ωI t2] cos[ωS 2 in0]
Ix
-->
IzSx cos[ωS 1 in0]
-->
Ix cos[ωI t2] cos[ωS 1 in0]
Polarization transfer
Acquisition
Polarization transfer
Chemical shift evolution ( n increments)
Chemical shift evolution
1H
15N
t1
t2
Polarization transfer: - relaxation (T1, T2)
- incomplete transfer
- other active couplings (pathways)
- many pulses (rf inhomogenity)
4
Chemical shift evolution
1H
15N
t1
t2
Polarization transfer: - relaxation (T1, T2)
- incomplete transfer
- other active couplings (pathways)
- many pulses (rf inhomogenity)
Chemical shift evolution: - relaxation (T1, T2)
- active couplings (unwanted pathways)
- 1.41 signal loss due to complex acquisition
- resolution limitation in 3D and 4D exp
( n increments per indirect dimensions)
TROSY:
Transverse Relaxationoptimized Spectroscopy
5
CSA AND DD contribution to
relaxation
6
[15N,1H]-TROSY
7
[15N,1H]-TROSY of DHNA
(110 kDa)
8
Magnetic field dependent
effect of TROSY
Magnetic field-dependent
effect of [15N,1H]-TROSY
100 kDa
30 kDa
9
TROSY is a building block
• 15N-resolved [1H,1H]-NOESY
• Triple resonance experiments
• Relaxation measurements
TROSY-Triple resonance
experiments
10
TROSY-HNCA (110 kDa DHNA;
Salzmann et al)
ZQ TROSY and 13Carom/methyl
TROSY
• TROSY can be expanded to transverse
relaxation optimization due to CSA/CSA cross
correlation: ZQ TROSY
• TROSY can be expanded to 13Cmethyl/arom due
to DD/DD(CSA) cross correlation
• TROSY can be applied to DNA/RNA for the
sugar 13C and base 15N
11
NMR of
large proteins
•
•
•
•
TROSY (up to 100 kDa)
CRINEPT (> 100 kDa)
H/D exchange (up to X MDa)
Membrane Proteins
2H,15N-labeled
GroEL (880 kDa)
[15N,1H]-TROSY
[15N,1H]-CRIPTTROSY
10
9
8
12
High resolution solution NMR of
macromolecules with a molecular weight up to 1
MDa
• Introduction
• Technique
– Polarization transfer
– T1(1H): inter-scan delay and water-protein interaction
– [15N,1H]-correlation experiments
• Application
– 2H,15N-labeled GroES in complex with GroEL
– 2H,15N-labeled GroEL in complex with GroES
[15N,1H]-TROSY
1H
15N
t1
t2
PFG
Transverse relaxation-optimization during polarization transfers
13
Polarization transfer: INEPT
y
x
1H
15N
Ix
2IySz
2IzSx
Polarization Transfer efficiencies
14
Polarization transfer: CRIPT
(Cross-correlated relaxation-induced
polarization transfer)
y
y
1H
15N
Ix
2IxSz
2IzSx
=
+
CRIPT Transfer Time T
0.02
250
200
SR1
I rel
0.015
100
T[s]
50
GroEL
0.01
DHNA
20C
0.005
2
8
T[ms]
46
GroES + GroEL
DHNA
4C
SR1
50
100
150
GroES + SR1
200
250
GroEL
300 τc[ns]
15
Polarization Transfer efficiencies
Polarization transfer: CRINEPT
(Cross-correlated relaxation-enhanced
polarization transfer)
y
φ
1H
15N
Ix
2IySz
2IzSx
+ evolution
16
Improvement of CRINEPT
by Glaser/Pervushin
φ
y
1H
15N
Ix
2IySz
2IzSx
Selective inversion
Polychromatic inversion
[15N,1H]-TROSY
1H
15N
t2
t1
PFG
[15N,1H]-correlation
experiments
TROSY
CRINEPT-TROSY
P t1 t2 N. of
T
PT
-
+ + 3
PT type
peak
selectio
n
3J
HN
CRINEPT-HMQC
CRIPT-TROSY
17
[15N,1H]-CRINEPT-TROSY
T
1H
T
T
t1
15N
t2
PFG
[15N,1H]-correlation
experiments
TROSY
CRINEPT-TROSY
P t1 t2 N. of
T
PT
+
+ + 3
+ + 2
PT type
peak
selectio
n
3J
HN
3J
HN+R C
CRINEPT-HMQC
CRIPT-TROSY
[15N,1H]-CRINEPT-HMQC-1H-TROSY
1H
15N
t2
t1
PFG
[15N,1H]-correlation
experiments
P t1 t2 N. of
T
PT
PT type
TROSY
CRINEPT-TROSY
+
+ + 3
+ + 2
3J
CRINEPTHMQC/TR
CRIPT-TROSY
+
-
3J
+ 2
peak
selectio
n
HN
3J
HN+R C
HN+R C
18
[15N,1H]-CRIPT-TROSY
1H
t2
t1
15N
PFG
[15N,1H]-correlation
experiments
P t1 t2
T
N. of
PT
PT type
TROSY
CRINEPT-TROSY
+
+ + 3
+ + 2.5
3J
HN
3J +R
HN
C
CRINEPTHMQC/TR
CRIPT-TROSY
+
-
3J +R
HN
C
+
+ + 1
+ 2
peak
selectio
n
RC
[15N,1H]-CRINEPT-TROSY of 2H,15N-labeled
GroES in complex with SR1 (0.5 MDa)
19
[15N,1H]-CRIPT-TROSY of 2H,15N-labeled
GroES in complex with SR1 (0.5 MDa)
ω1(15N)
[ppm]
ω2(1H) [ppm]
[15N,1H]-CRINEPT-HMQC-1H-TROSY of 2H,15Nlabeled GroES in complex with SR1 (0.5 MDa)
20
2H,15N-labeled
GroEL (880 kDa)
[15N,1H]-TROSY
[15N,1H]-CRIPTTROSY
10
9
8
High resolution solution NMR of
macromolecules with a molecular weight up to 1
MDa
• Introduction
• Technique
– Polarization transfer
– [15N,1H]-correlation experiments
– T1(1H): inter-scan delay and water-protein interaction
• Application
– 2H,15N-labeled GroES in complex with GroEL
– 2H,15N-labeled GroEL in complex with GroES
21
Water-Protein Interaction
T1(1H)
[s]
10000
100
SR1 in D2O
1
BPTI
0.01
10-12
τc [s]
10-9
T1H2O
10-8
T1H2O = T1D2O + ρHOH+ σHOH
10-7
10-6
Fast exchange
22
High resolution solution NMR of
macromolecules with a molecular weight up to 1
MDa
• Introduction
• Technique
– Polarization transfer
– [15N,1H]-correlation experiments
– T1(1H): inter-scan delay and water-protein interaction
• Application
– 2H,15N-labeled GroES in complex with GroEL
– 2H,15N-labeled GroEL in complex with GroES
GroES-GroEL interaction
Sequential backbone assignment of
2H, 13C,15N-labeled GroES with
TROSY-based triple resonance exp.
Homo heptamer
70 kDa
[15N,1H]-CRIPT-TROSY
Of 2H,15N-labeled GroES
In complex with 2H-labeled
SR1 or 2H-labeled GroEL
Homo heptamer
500 kDa or 950 kDa
23
2H, 15N-labeled
free
GroES
in complex with SR1
114.0
116.0
ppm
ppm
118.0
9.5
9.0
9.5
9.0
ppm
9.8
9.8
ppm
[15N,1H]-CRIPT-TROSY
CRIPT Transfer Time T -> τc
0.02
250
200
SR1
I rel
0.015
100
T[s]
50
GroES + SR1
DHNA
20C
0.005
GroES + GroEL
GroEL
0.01
2
8
T[ms]
46
GroES + GroEL
DHNA
4C
SR1
50
100
150
GroES + SR1
200
250
GroEL
300 τc[ns]
24
GroES
GroES
GroES + SR1
GroES + SR1
GroES
GroES
GroES + SR1
GroES + SR1
GroES+GroEL
GroES+GroEL
GroES+GroEL
GroES+GroEL
25
Assignment
26
318
256
310
213
214
ω1(15N)
[ppm]
326
361
ω2(1H) [ppm]
GroES-GroEL complex
27
Glu 310
Conclusion
Identification of protein-protein interfaces is
possible with molecular complexes of 1 MDa
Prerequisites:
• 2H,15N-labeling
• Multimeric proteins or highly soluble proteins
• Partial sequential assignment
28
NMR of
large proteins
•
•
•
•
TROSY (up to 100 kDa)
CRINEPT (> 100 kDa)
H/D exchange (up to X MDa)
Membrane Proteins
Inclusion Bodies of E. Coli
29
Structural Studies of inclusion bodies of BMP2 and ESAT6
Bone morphogenetic protein-2
(BMP2) is a member of the
transforming growth factor beta
(TGF-beta) superfamily, and it
plays an important role during
early stages of embryonic
development.
6-kDa early secretory antigenic
target (ESAT6) is an
immunogenic antigen
recognized during the
Tuberculosis disease.
Thio-T binding indicates inclusion bodies are amyloidlike
ThT binding
20
15
10
5
0
BMP2
ESAT6
MOG
_-Synuclein
30
Quenched H/D exchange by NMR
D2O
H2O
N-H
N-H
N-H
DMSO
N-H
N-D
N-D
Hoshino, Goto et al., 2002
31
H/D exchange of BMP2
Inclusion Body Study
Lei Wang
4.24.2007
H/D exchange of BMP2 inclusion bodies
1
V67
Irel
I62
V21
F41
0
0
100
H/D exchange Time [hours]
32
H/D exchange of BMP2 inclusion bodies
Amyloid Fibrils
•
•
•
•
•
•
Disease-associated
Infectious proteins (prions)
Native functions of fibrils
Protein structure dogma
Inherent to all proteins (Dobson)
Nanotechnology (storage container, in
vivo mini-column)
33
NMR investigations on KcsA
potassium channel
20 pA
500 ms
1s
7 -> 4 pH
Roland Riek
The Salk Institute
KcsA x-ray structure
(MacKinnon et al,
1998)
34
KcsA x-ray structure
(MacKinnon et al,
1998)
KcsA x-ray structure
(MacKinnon et al,
1998)
35
KcsA x-ray structure
(MacKinnon et al,
1998)
KcsA x-ray structure
(MacKinnon et al,
1998)
36
KcsA x-ray structure
(MacKinnon et al,
1998)
Goooooooooal !!!
KcsA (4*160 aa, 130 kDa)
17 Gly
17 Gly peaks
(plus 3 sharp
little peaks)
17 Arg
17 Arg peaks
5 Trp
5 Trp peaks
(plus 2 sharp
satelite peaks)
37
Technical comments
KcsA binds agitoxin 2 and is a
tetramer
38
Selective Labeling and Alaselective 2D HNCA
15N-Asp
15N-Xxx
15N-Phe
sequential to Ala
115.00
116.00
118.00
118.00
120.00
120.00
120.00
122.00
122.00
125.00
124.00
8.50
8.00
124.00
8.50
7.50
8.00
7.50
9.00
8.50
8.00
7.50
Leu, Phe, Tyr, Asp, Ile
Specific Assignment
Tyr78
Tyr82
Tyr62
Tyr137
ω 1(15N)
[ppm]
120
Tyr45
120
9
8.5
8
ω 2(1H) [ppm]
125
9
8
7.5
KcsA(F78Y)
39
9
1
0
9
1
1
3
1
1
7
1
2
1
1
2
5
1
2
9
1
3
3
1
3
7
1
4
1
1
4
5
1
4
9
1
5
3
1
5
7
5
1
7
3
9
9
9
8
8
5
8
7
7
3
7
6
5
1
7
6
6
5
3
9
5
4
5
1
7
4
4
3
0
1
1
9
3
3
2
9
9
13
3
13
7
14
1
14
5
14
9
15
3
15
7
12
12
5
11
3
11
7
12
1
10
1
10
5
10
9
97
93
89
85
81
77
73
69
65
61
57
53
49
45
41
37
33
29
25
21
17
13
6
0
1
7
5
1
1
2
2
9
deltaRC
6
1
1
6
12
1
12
6
13
1
13
6
14
1
14
6
15
1
15
6
11
11
1
10
6
10
96
91
86
81
76
71
66
61
56
51
46
41
36
31
26
21
16
11
-1
3
1
1
5
deltaRC
pH 7.0
closed
5
pH 4.0
open
1
deltaCA[ppm]
KcsA assignment
6
Ca Chemical Shifts
5
4
3
2
1
0
-2
5
Residue
4
3
2
1
-1
0
-2
0.6
0.8
1
Residue
-0.4
-0.2
-0.6
0.4
-0.8
0.2
-1
0
Residue
40
Detergent-KcsA interactions
Hydrogen exchange of KcsA
41
9
5
7
1
3
0
0
1
9
7
3
9
2
1
1
2
5
1
2
9
1
3
3
1
3
7
1
4
1
1
4
5
1
4
9
1
5
3
1
5
7
1
1
1
5
9
8
8
8
7
7
7
3
6
9
5
1
6
6
7
3
5
5
9
4
1
1
1
4
5
4
7
3
3
3
9
5
2
1
0
1
7
2
2
1
1
9
10
3
7
12
1
12
5
12
9
13
3
13
7
14
1
14
5
14
9
15
3
15
7
11
5
9
10
11
1
10
97
93
89
85
81
77
73
69
65
61
57
53
49
45
41
37
33
29
25
21
17
13
6
1
9
deltaRC
6
1
96
10
1
10
6
11
1
11
6
12
1
12
6
13
1
13
6
14
1
14
6
15
1
15
6
91
86
81
76
71
66
61
56
51
46
41
36
31
26
21
16
11
-1
3
1
1
5
deltaRC
pH 7.0
closed
5
pH 4.0
open
1
deltaCA[ppm]
Ca Chemical Shifts
6
5
4
3
2
1
0
-2
5
Residue
4
3
2
1
-1
0
-2
0.6
0.8
1
Residue
-0.4
-0.2
-0.8
-0.6
0.2
0.4
-1
0
Residue
pH-Titration of 15N-Tyr KcsA
42
Population
in open state
75%
15N
50%
25%
Tyr78
0%
pH
43
CT-ZQ-TROSY for the measurement of 3J(N,HA) couplings
44
More Dynamics?
- Fast exchange between open and closed state ->
inactivated state
-19F-Tyr good probe to measure dynamics for large
proteins (high sensitivity, CSA dominates)
- Dynamical change:
pH 7 (closed): slow dynamics present
pH 4 (open): slow dynamics absent
3000
pH 7: T2(19F-Y78) &
T1ρ(19F-Y78)
2500
2500
2000
pH 4: T2(19F-Y78) &
T1ρ(19F-Y78)
2000
1500
Irel
1500
1000
1000
500
500
0
0
0.002
0.004
-
0.006
0.008
0.01
0.012
0.014
0
0
0.002
T [s]
0.004
0.006
0.008
0.01
T [s]
NMR of
large proteins
•
•
•
•
TROSY (up to 100 kDa)
CRINEPT (> 100 kDa)
H/D exchange (up to X MDa)
Membrane Proteins
45
NMR of
large proteins
• TROSY: Konstantin Pervushin, Gerhard
Wider, Kurt Wuthrich, Jocelyne Fiaux
• H/D exchange: Thorsten Luhrs, Marcial
Vilar, Lei Wang
• Membrane Proteins: Kent Baker,
Christos Tzitzilonis, Senyon Choe
46