- EPJ Web of Conferences

EPJ Web of Conferences 30, 03002 (2012)
DOI: 10.1051/epjconf/20123003002
© Owned by the authors, published by EDP Sciences, 2012
Experiment and Modelling in Structural NMR
November 28th – December 2nd 2011
INSTN – CEA Saclay, France
Nicolas Birlirakis
ICSN CNRS, France
3D modern techniques
[03002]
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/20123003002
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which
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Lectures

ORGANISATION
History, General principles
M. Goldman, CEA Saclay
Formalism and Interactions
H. Desvaux, CEA Saclay
Molecular Modelling
M. Nilges, Institut Pasteur
Anisotropy for Conformational studies
M. Blackledge, CEA Grenoble
Oriented media
D. Merlet, Univ. Paris-Sud 11
Techniques of solid-state
NMR
D. Massiot, CNRS Orléans
NMR crystallography
F. Taulelle, UVSQ
•
Coordinator for the INSTN - CEA Saclay
Constantin MEIS, INSTN/DIR
•
Coordinators for the CEA
Patrick BERTHAULT, DSM/IRAMIS
Thibault CHARPENTIER, DSM/IRAMIS
•
Coordinators for the Universities
Pierre Richard DAHOO, Versailles Univ., UVSQ
Francis TAULELLE, Versailles Univ., UVSQ
Eric SIMONI University Paris-Sud 11
Winter-School
“Experiment and Modelling in
Structural NMR”
Denis MERLET, University Paris-Sud 11
3D Modern Techniques
N. Birlirakis ICSN - CNRS
Interactions ligandmacromolecules
G. Lippens, University of Lille
Bio-Solid State NMR
H. Oschkinat, University of
Berlin
Bioinformatics-protein
assembly
R. Guérois, CEA Saclay
Materials Modelling &
Simulations
C. Meis, INSTN - CEA, Saclay
DFT calculations of NMR
parameters.
J. Yates, Oxford University
Inorganic MaterialsCeramics
S. Ashbrook, University of
St Andrews
•
Secretary, Information and Registration
Dominique PARISOT,
INSTN - CEA Saclay
Bat. 395 - PC N°35
Gif-sur-Yvette F-91191
: +33 1 69084882
email: [email protected]
A limited number of scholarships can be
obtained for PhD students
contact : [email protected]
Amorphous materials
T. Charpentier, CEA Saclay
Hyper-polarization
G. De Paepe, CEA Grenoble
Fast NMR
N. Giraud, Univ. Paris-Sud 11
NMR Diffusometry :
Dynamics in Complex Systems over many decades
R. Kimmich, University of Ulm


The aim of the school is to provide basic understanding on the
coupling of theoretical Modelling and Simulation methods to the
Experiments in structural NMR, for biology and material science.
It is composed of Seminars in the morning and Practical Training in the afternoon involving both the theoretical and experimental aspects.
November 28 - December 2, 2011
INSTN - CEA, Saclay
http://www-instn.cea.fr (events 2011)
3D NMR experiments applied to proteins
H
H
+
H3N
C*
COO-
+H N
3
R
amino acid
C
C
R1
O
H
H
N
C
C
R2
O
H
H
N
C
COO-
Rn
peptide < 50aa < protein
Essential constituents of all living organisms
Linear chains consisting of 20 L-amino acids
Their folding to particular 3D structures triggers their
biological function
Attractive targets for therapeutic applications once their
structure-activity relationship is understood
03002-p.2
NMR studies of peptides (<50aa)
General considerations
- Chemically synthesized molecules (SPPS)
- Isotopic labelling expensive → homonuclear 1H-1H 2D experiments
Spectral assignment
- Starting point for magnetisation transfer: backbone NH protons (experiments in H2O)
- Spin system identification via scalar couplings: 2D COSY, RELAYS, TOCSY
- Sequential assignement: 2D NOESY
Extraction of geometrical informations
- Dihedral angle φ determination from 3JHN-Hα couplings: 2D-COSY-DQF (Karplus equations)
- Interproton distance determination: 2D NOESY (build up with several τm)
P
E
COSY
t1
RELAY
t1
TOCSY
t1
NOESY
t1
M
[
1/4J
A
1/4J
]
antiphase transfer
H
H
N
C
C
H
H
O
N
C
C
CH3 O
n
CH
H 3C
CH3
in-phase transfer
CSL
τm
t1
ROESY
SL
NMR studies of small proteins (50-100aa)
General considerations
- Favorable T2 relaxation
- Increased 1H signal density → increased spectral complexity and peak overlapping (structuration dependent)
Spectral assignment and structure elucidation
- Same strategy as previously
- Addition of one more dimension to increase resolution: first 3D experiment NOESY-TOCSY, Nature 332, 374 (1988)
P
E1
M1
E2
t1
τm
t2
M2
A
Breakthrough
- Recombinant protein expression → affordable 15N labelling (natural abundance 0.366%)
- The gene encoding the protein of interest is inserted to the plasmids
- Plasmids contain genes confering resistance to antibiotics (selection)
- Plasmids are inserted to the bacteria (transformation)
- Use of rich media (LB) for cell replication
- Induction in poor media (M9) containing 15NH4Cl
Escherichia coli
- Rare codons
- Protein folding and inclusion bodies
- Post-translational modifications
03002-p.3
NMR studies of small proteins (50-100aa)
Heteronuclear 3D experiments
- Resolve the 1HN overlapping by the use of 15NH frequency : 4ppm versus 30-35ppm
Sensitivity considerations (S/N)
S ~ n A (1/T) Bo3/2 γobs3/2 γexc T2
Cryoprobe technology: N↓
√Nscan
n: sample quantity (v*c)
A: active nuclei abundance
NOESY(TOCSY) - fHSQC
φ1
[
t1
1H
1H
j
]
τm
y
1/4J
WG
1/4J
/ 1H i
1/4J
1/4J
t3
1HN
φ2
t2
15N
15NH
TOCSY mixing: use clean CSL
schemes to suppress dipolar
cross relaxation and NOESY/
ROESY artifacts
i
cpd
i
Gr
Going from 2D to 3D
3D NOESY-HSQC H-H-N (τm=150ms)
8 scans
2D NOESY (τm=150ms)
4 scans
ppm
0
F2 [ppm]
2
110
4
125
120
]
ppm
F1 [
10
8
6
4
2
0
ppm
03002-p.4
130
8
10
6
8
115
6
10
9
8
7
F3 [ppm]
3D data analysis
clean TOCSY-HSQC (tm=70ms)
or ROESY-HSQC
([email protected]°)
NOESY-FHSQC (tm=150ms)
A116QB-E113N-H
A112QB-E113N-H
E113HB#-N-H
2
2
E113HG3-N-H
E113HG2-N-H
E113HA-N-H
ω1 - 1H (ppm)
A112HA-E113N-H
G110HA3-E113N-H
4
4
G110HA2-E113N-H
M109HA-E113N-H
6
6
F114H-E113N-H
L115H-E113N-H
G110H-E113N-H
8
?
9.0
8.5
7.5
8.0
7.0
9.0
8.5
8
?
A112H-E113N-H
?
8.0
7.5
7.0
9.0
8.5
8.0
7.5
7.0
ω3 - 1H (ppm)
Heteronuclear correlation building blocks
refocused HSQC (JSS, T2S)
HMQC (JII, JSS, T2I, T2S)
y
1H
1/2J
-Iy
t2
1/2J
2Ix,ySz
2Ix,ySz
1H
Iy
1/4J
-Iy
φ1
1/4J
1/4J
cpd
2IxSz
1/4J
1/4J
1/4J
1/4J
2IxSz
Iy
t2
φ1
t1
15N
1/4J
cpd
-2Ix,ySy
15N
t1
-2IzSy
cpd
Sx
HSQC (JSS, T1I, T2S)
y
1H
1/4J
-Iy
1/4J
1/4J
2IxSz
2IySz
2IySxS
φ1
15N
t1
-2IzSy
1/4J
Axial peak suppression: φ1 and rec cycled x, -x
t2
Quadrature detection at the indect dimension
using φ1 incrementation:
-Ix
2IySx
TPPI (axial peak at the edges, folding)
States (axial peak in the middle, aliasing)
States-TPPI (axial peak at the edges, aliasing)
cpd
-2IzSy
-2IzSx
03002-p.5
Heteronuclear correlation building blocks
refocused HSQC (JSS, T2S)
HMQC (JII, JSS, T2I, T2S)
y
1/2J
-Iy
t2
1/2J
2Ix,ySz
2Ix,ySz
1H
Iy
1/4J
-Iy
1/4J
φ1
cpd
-2Ix,ySy
-2IzSy
-Iy
1/4J
1/4J
2IxSz
2IySz
2IySxS
t1
-2IzSy
1/4J
1/4J
2IxSz
Iy
t2
cpd
Sx
y
φ1
15N
1/4J
sensitivity enhanced HSQC (JSS, T1I, T2S)
y
1/4J
1/4J
cpd
t1
15N
HSQC (JSS, T1I, T2S)
1H
1/4J
φ1
t1
15N
1/4J
2IxSz
1/4J
t2
-Ix
2IySx
1/4J
-Iy
1/4J
1/4J
15N
1/4J
1/4J
t2
1/4J
2IxSz
φ1
cpd
-2IzSy
-2IzSx
1H
y
±y
t1
-2IzSy
cpd
-2IzSy 2IySz
-2IzSx 2IySx
-Ix Iz
-Iz
2IySx ±2IySz ±2IySz
Iy
Ix
±
1H
SUMMATION → 2Iy
DIFFERENCE → 2Ix
Increase S/N : √2 (neglecting relaxation)
NMR studies of medium size proteins (100-200aa)
- Due to increased spectral complexity, peak assignement is difficult through
3D H-H-N TOCSY/NOESY-HSQC experiments
- Slow molecular tumbling results in rapid T2 proton relaxation: COSY and TOCSY
HN → Hα magnetisation transfer fails especially in helical regions (3JHN-Hα ~ 3Hz)
03002-p.6
NMR studies of medium size proteins (100-200aa)
- Recombinant expression of 13C, 15N labelled proteins using 15NH4Cl and 13C6-glucose
→ access to triple-resonance experiments
H
90Hz
Hα
H
140Hz
90Hz
Cα
N
35Hz
55Hz
C
15Hz
N
7Hz
Cβ
Hα
140Hz
11Hz
Cα
55Hz
C
<1Hz
Cβ
O
i-1
O
i
- Resolve the 1HC overlapping by using 13CH frequency: 5ppm versus 65-70ppm
for the aliphatics
- Use of heteronuclear couplings 1H-15N, 1H-13C, 13C-13C, 15N-13C (1J and 2J) for
unambiguous intra- and inter-residual assignment
- Magnetisation transfers implicating slowly relaxing nuclei
Backbone assignment via out and back 3D experiments
HNCO (100)
H
N
Hα
H
Cα
C
i-1
O
N
HN(CA)CO (13/4)
Hα
H
Cα
C
i
O
N
Hα
Cα
C
i-1
O
HNCA (50/15)
H
N
Hα
H
Cα
C
i-1
O
N
N
Hα
H
Cα
C
Cβ
O
i-1
N
N
Hα
Cα
C
i
O
HN(CO)CA (71)
H
Hα
Cα
C
i
O
N
Hα
C
i-1
O
H
C
Cβ
O
N
Hα
Cα
C
i
O
HN(CO)CACB (13-9)
Hα
Cα
H
Cα
HNCACB (4-1.7/1.3/0.5)
H
H
N
i
Hα
Cα
C
Cβ
O
i-1
03002-p.7
H
N
Hα
Cα
C
Cβ
O
i
The HNCA experiment
y
1
15
H (I)
∆1
y
-x
∆1 2∆1
2∆1 ∆1
WALTZ-y
-Iy 2IxSz
y
∆2
N (S)
∆2−t1/2
-2IzSy
2SyTz
t2/2
13
t1/2
2SyTz
y
φ2
∆1
-Ix
2IySz
y
φ1
∆2
WG
∆2
CPD
-2IzSy
t2/2
Cα (T)
-2SzTy
CO
13
BS BS
Gz
T:+1
T:0
T:-1
T:+1
T:0
T:-1
T:+1
T:0
T:-1
S:+1
I:+1
S:0
S:-1
T:+1
T:0
T:-1
T:+1
T:0
T:-1
T:+1
T:0
T:-1
S:+1
I:0
S:0
S:-1
T:+1
T:0
T:-1
T:+1
T:0
T:-1
T:+1
T:0
T:-1
S:+1
I:-1
S:0
S:-1
The HNCA experiment
y
1
H
∆1
y
-x
∆1 2∆1
2∆1 ∆1
WALTZ-y
HNi
N
φ1
y
∆2
15
NHi
t2/2
t1/2
15NH
y
φ1
∆1
1HN
y
∆2−t1/2
∆2
WG
t2/2
∆2
i
CPD
i
Cα
13
13Cα
CO
13
i,i-1
BS BS
Gz
Water magnetization at z prior to WATERGATE module
Proton decoupled version (longer T2 relaxation for 15N, 13C nuclei)
Constant time at 15N dimension
1
JCαCβ couplings not refocused during t2
∆1 = 1/41JHN = 2.7m
∆2 = 12m (relaxation optimized, 1/4 JNCα ~ 22m)
Same sequence for HNCO experiment (via Cα-CO channel interconversion)
03002-p.8
Experimental aspects of triple
resonance experiments
- Shaped pulses
- Pulsed field gradients
- Water suppression
Hard pulses
Excitation profile of a rectangular
90° pulse τp = 1ms
1/τp
B1
τp
t
600
θ
X
B1
-600
sinc (=sinx/x)
Z
Flip angle θ
depends on γB1τp
0
Y
15000
10000
75
50
5000
25
0
-5000
-10000
-15000
0
-25
-50
-75
Hz
ppm
(DRX-800, 13C)
Rectangular hard pulses (polychromatic): γB1/2π >>> ∆F
Typical hard pulse lengths @ 800MHz, max admitted power:
1H → 7-...µs
15N → 39.5µs
13C → 14µs
03002-p.9
Shaped selective pulses
Amplitude modulation: first trials
Rectangle
Sinc
Triangle
Amplitude
Amplitude
Amplitude
Phase
Phase
Phase
200
3000
400
2000
1000
600
0
800
-1000
-2000
200
[points]
-3000
3000
Hz
400
2000
600
1000
90°
0
800
-1000
-2000
200
[points]
-3000
3000
Hz
400
2000
1000
600
0
-1000
800
-2000
[points]
-3000
90°
90°
Shaped selective pulses
Amplitude modulation: peak selection
Gaussian_1
Gaussian_1
Gaussian_1
Amplitude
Amplitude
Amplitude
Phase
Phase
Phase
200
3000
2000
400
1000
600
0
90°
-1000
800
-2000
-3000
200
[points]
Hz
3000
400
2000
1000
600
0
800
-1000
-2000
270°
-3000
200
[points]
3000
Hz
400
2000
1000
0
180°
180°y, -y
90° Gaussian δ and J evolution during pulse:
600
270°
90°x
τp
τp
03002-p.10
τp/2
-1000
800
-2000
-3000
[points]
Hz
Hz
Shaped selective pulses
Amplitude/phase modulation: band selection (top-hat profile)
Gaussian Cascade and BURP famillies
Cascade Q3
Cascade Q5
U-BURP 2
Amplitude
Amplitude
Amplitude
Phase
Phase
Phase
200
6000
400
4000
2000
600
0
90°
800
-2000
-4000
-6000
[points]
Hz
200
6000
400
4000
2000
600
0
-2000
800
-4000
-6000
200
[points]
Hz
6000
4000
400
2000
180°
600
0
-2000
800
-4000
-6000
[points]
Hz
90°
Shaped selective pulses
Amplitude/phase modulation: band selection (top-hat profile)
Gaussian Cascade family
- Universal pulses: Q5 (90°, 6.18) and Q3 (180°, 3.41/3.448)
- Excitation pulse: G4 (7.82)
- Inversion pulse: G3 (3.556)
BURP famillies
- Universal pulses: U-BURP (90°, 4.666) and RE-BURP (180°, 4.64/5.814)
- Excitation pulses: E-BURP1 (4.514) and E-BURP2 (4.952)
- Inversion pulses: I-BURP1 (4.498) and I-BURP2 (4.53)
Figure of merit (depends on the shape):
FM = τp∆Ω
Frequency shifted (laminar) pulses : ∆φ = 2π τp ∆F / k
03002-p.11
Examples of selective excitation
Advantages
- of a spectral region
• zoom into a precise spectral
region
• suppression of indesired
signals (solvent)
• speed-up experiments
• resolution increase
- precise measurement of δ et J
- refocusing of homonuclear
couplings
• posibility to direct transfers
• possibility to treat an homonuclear
system as heteronuclear
- of several peaks
- of two peaks
- of one peak
1D spectrum of
strychnine in CDCl3
8.0
7.0
6.0
5.0
4.0
3.0
2.0
ppm
Examples of 2D selective experiments
90°
classical NOESY
90°
t1
90°
t2
tm
F1
1D selective NOESY
90°
90°
90°
t1
90°
tm
90°
t2
semi-soft NOESY
F2
90°
soft NOESY
90°
t1
03002-p.12
tm
90°
t2
90°
tm
t2
Selective excitation in triple resonance experiments
- Differentiation between 13Cα (or 13Caliph) and 13CO regions
Use of rectangular pulses: τ180° = √3 / 2∆Ω and τ90° = √15 / 4∆Ω
where ∆Ω is de difference of pulse carrier and the first null point
Problems: inhomogenous excitation/inversion and cryoprobe arcing if executed
simultaneously high power 13C and 15N pulses
Use of universal top-hat pulses (or interleaved G4 and G4tr)
90° pulse comparison: 36µs square and 300µs G4
180° pulse comparison: 33µs square and 200µs Q3
0.80
0.80
0.60
0.60
0.40
0.40
0.20
0.20
-0.00
-0.00
-0.20
CO
-0.20
-0.40
Caliph
CO
-0.60
-0.40
Caliph
-0.60
-0.80
30000 20000 10000
0
-10000 -20000 -30000
ATTENTION
Offset-dependent
transient Bloch-Siegert
phase shifts in
homorefocusing
-0.80
Hz
30000 20000 10000
0
-10000 -20000 -30000
Hz
- Selective perturbation of 1HN region (BEST experiments)
- Solvent selection (flipback or suppression)
Broadband inversion and decoupling
- High field spectrometrs → large 13C spectral width in Hz → imperfect inversion/decoupling
→ use of adiabatic pulses:
smoothed CHIRP and WURST families
180° pulse comparison: 28µs square and 500µs CA-WURST-4
0.80
Adiabatic decoupling:
0.60
0.40
0.20
-0.00
-0.20
Caliph+arom
-0.40
-0.60
-0.80
30000 20000 10000
0
-10000 -20000 -30000
Use P4M5 supercycle
Use bi-level decoupling for reduction
of sidebands in 1H-1H-13C and 1H-1H-15N
experiments
Homonuclear decoupling → offset
dependent Bloch-Siegert frequency shifts
Hz
- Adiabatic 15N inversion and decoupling is used to reduce power and thus probe heating,
important in rapid scan conditions
- Adiabatic pulses are tolerant to Rf inhomogeneity
- Interesting pseudoadiabatic pulses: Bip and power-BIBOP for inversion and power-BEBOP
for excitation
03002-p.13
Pulsed field gradients
Linear variation of B0 at a certain direction
- Coherence transfer pathway selection:
- Artifact suppression per scan ie
1H12C
Σ γi pi Gi = 0
artifacts with short phase cycling (...substraction)
- Solvent suppression and control of water magnetisation (radiation damping)
- Translational diffusion measurements
- Magnetic Resonance Imaging
gradient sensitivity enhanced HSQC (JSS, T1I, T2S)
y
1H
1/4J
y
1/4J
1/4J
φ1
1/4J
1/4J
t2
1/4J
±y
t1
15N
cpd
80
±8
Water suppression
Strong water (~50M) signal deteriorates spectral dynamic range
High fields + cryoprobes → strong radiation damping → large water signal
Strategies
- Observe an heteroatom
- Presaturation: forbitten in the case of fastly exchangable protons
- Coherence selection with gradients
- Return of water at z axis: polynomial sequences (jump and return...), water selective flip-back
pulses, manipulation of radiation dumping
- Dynamic filters: based on the differences in relaxation rates or diffusion coefficients
- Selective defocusing of water via a gradient echo (WATERGATE, excitation sculpting)
- Purging pulses: B0 or Rf gradient
y
90°y
-y
1H
90°y
1H
15N
Gr
Gr
03002-p.14
1/4J
1/4J
-Iy
-Iy
2IxSz
Iy
SLx
2IzSz
Iy
φ1
The HN(CA)CO experiment
y
1
H
∆1
y
-x
∆1 2∆1
2∆1 ∆1
WALTZ-y
HNi
15
y
∆2
N
∆2−t1/2
NHi
13
13
Cα
13Cα
∆3
∆3
∆2
15NH
∆3
i
CPD
i
BS
i,i-1
y
φ2
t2/2
CO
1HN
t1/2
y
y
∆3
∆1
y
φ1
∆2
WG
t2/2
BS
BS
13CO
i,i-1
Gz
INEPT version
∆1 = 1/41JHN = 2.7m
∆2 = 12m (relaxation optimized, 1/4 JNCα ~ 22m)
∆3 = 3.5-4m (relaxation optimized, 1/4 JCOCα = 4.5m)
The HN(CO)CA experiment
y
1
H
∆1
y
-x
∆1 2∆1
2∆1 ∆1
WALTZ-y
HNi
15
N
y
∆2
∆2−t1/2
NHi
13
CO
Cα
13
t1/2
15NH
∆3
13CO
i,i-1
∆3
BS
y
φ2
t2/2
∆2
i
13CO
i,i-1
t2/2
BS
13Cα
i,i-1
(DQ)
Gz
mixed INEPT / DQ version
∆1 = 1/41JHN = 2.7m
∆2 = 13.5m (relaxation optimized, 1/4 JNCO = 33m)
∆3 = 8m (relaxation optimized, 1/2 JCOCα = 9m)
03002-p.15
∆1
1HN
y
φ1
∆2
WG
i
CPD
The HNCACB experiment
y
-x
∆1 2∆1
2∆1 ∆1
y
1
∆1
H
WALTZ-y
HNi
φ1
y
∆2
N
15
NHi
φ3
φ1
∆3
13Cα,β
i,i-1
t2/2
∆3
1HN
t1/2
∆2
15NH
y
y
t2/2
∆3
∆1
y
∆2−t1/2
∆2
WG
∆3
i
CPD
i
Cα
13
13Cα
13Cα
i,i-1
CO
13
i,i-1
BS
BS
Gz
mixed INEPT / RELAY sequence
∆1 = 1/41JHN = 2.7m
∆2 = 12m (relaxation optimized, 1/4 JNCα ~ 22m)
∆3 = 3.6m (relaxation optimized, 1/4 JCαCβ = 7.1m)
1
JCαCβ and 1JCβCγ couplings not refocused during t2
Cα, Cβ peaks of opposite sign
Rapid pulsing - BEST experiments
- In 1H-15N correlation experiments 1HN longitudinal dipolar relaxation is accelerated
if the rest of the protein protons (aliphatics) and 1H2O are unperturbed (at z axis)
- Substitution of 1H hard pulses with shaped ones (top-hat ).
BEST-HSQC (JSS, T1I, T2S)
SOFAST-HMQC ( J SS, T2I, T2S)
y
1H
1/2J
1/2J
t2
1H
φ1
15N
1/4J
1/4J
1/4J
1/4J
t2
φ1
t1
cpd
15N
t1
: PC9 pulse (flip-angle 120°, Ernst angle)
band-selective excitation pulse
: PC9 pulse (flip-angle 90°)
band-selective 90° pulse
: band-selective universal 180° pulse
: band-selective universal 180° pulse
03002-p.16
cpd
Rapid pulsing - BEST experiments
SOFAST-HMQC
10
9
BEST-HSQC
8
7
105
105
110
110
115
115
120
120
125
125
130
130
ppm
10
90° pulse → 1770µs Q5.1000
1
180° pulse → 1260µs Q3.1000
9
8
7
ppm
H carrier frequency @ 7.999ppm
P5M4 15N decoupling using 2ms CA-WURST-2 adiabatic pulse
1scan, 128 transients, AQ=70ms, RD=300ms, Total Experimental Time: 55s
Fast out and back experiments: BEST-HNCA
y
∆1
1
H
∆1
y
∆1
2∆1
y
y
15
N
∆2
y
φ1
t1/2
∆2
∆1
∆2−t1/2
∆2
CPD
y
φ2
t2/2
t2/2
Cα
13
CO
13
BS
BS
Gz
: 180° broadband inversion pulse → 155µs power-BIBOP pulse
10kHz inversion, tolerant to 20% inhomogeneity
03002-p.17
3D spectra for some BEST out and back experiments
BEST-HNCO: 4scans, 58min
F2[ppm]
115
110
pm]
F1 [p
40
50
60
130
125
120
115
pm]
F1 [p
174
176
125
120
130
8
7
F3[ppm]
9
10
8
7
F3[ppm]
BEST-HN(CA)CO: 4scans, 59min
10
7
F3[ppm]
10
7
F3[ppm]
115
125
190
130
F1
170
]
[ppm
120
115
pm]
F1 [p
54
56
58130
125
120
8
8
110
F2[ppm]
110
F2[ppm]
110
115
120
125
9
9
BEST-HN(CO)CACB: 4scans, 4h 9min
F2[ppm]
9
180
130
pm]
F1 [p
110
F2[ppm]
110
115
pm]
F1 [p
55
60
65130
125
120
10
BEST-HN(CO)CA: 4scans, 2h
10
BEST-HNCACB: 4scans, 3h 54min
F2[ppm]
BEST-HNCA: 4scans, 1h 53min
9
8
7
F3[ppm]
10
9
8
7
Conclusions on BEST out and back experiments
- Usefull for protonated proteins < 150aa (above, fractional deuteration is required)
- Other fast methods are based on reduced sampling at the indirect dimensions:
Spectrum compression through folding
Exponential sampling and maximum entropy
Projection reconstruction (radial sampling)
Hadamard transform
Filter Diagonalisation Method
Sharc NMR
...
03002-p.18
F3[ppm]
Backbone and side chain assignment
via transfer 3D experiments
HBHA(CBCACO)NH
H
In H2O:
Hα
N
Cα
Hβ
O
H
C
N
Hα
Cα
(H)CC(CO)NH-TOCSY / H(CCCO)NH-TOCSY
O
H
C
N
Cα
Hβ
Cβ
Hγ
Cγ
Cβ
i-1
i
Hδ
Hα
O
H
C
N
Hα
Cα
O
C
Cδ
i-1
i
In D2O:
HACACO
H
N
Hα
HACACO-TOCSY
H
Cα
C
i-1
O
N
H
Hα
Cα
C
i
O
N
Hα
Cα
O
H
C
N
i-1
H(C)CH-TOCSY / (H)CCH-TOCSY
O
H
Cα
C
N
Cβ
Hα
Hα
H
C
N
Hα
O
Cα
C
Hβ
Cβ
Hβ
Cγ
Hγ
Cγ
Hγ
Cδ
Hδ
Cδ
Hδ
i
Cα
O
i-1
i
The HCCH-TOCSY experiment
δ → t1
J → 2∆1
H
1
∆1
y
∆1
t1/2 t1/2 ∆1
y
13
C
13CO
∆1/2
t2/2
∆1/2 t2/2
∆1
-y
FLOPSI
∆1/2
∆1/2
BS
Gr
purging
Refocused INEPT / z-TOCSY
Constant time at both indirect dimensions
∆1 = 1/21JHC = 37m (optimized for CH2)
Purging of residual water magnetization with B0 and RF gradients
03002-p.19
CPD
NMR studies of large proteins (>200aa)
- Decrease transverse 13CH relaxation rates through deuteration:
Expression in D2O → up to 70% deuteration (isotopic effect)
Expression in D2O+U[13C, D]-glucose → up to ~100% deuteration
Use of pyruvate or α-ketobutyrate/α-ketoisovalerate for production
of methyl protonated perdeuterated proteins.
(back-exchange of ND to NH, introduction of D decoupling)
- Decrease transverse 15N1H relaxation rates through TROSY effect at high field:
Selection of the slower relaxing component due to the destructive
interference of cross-correlated relaxation (DD - CSA)
Decoupled HSQC
Non-decoupled HSQC
TROSY
15 N
1H
- At high fields transverse 13CO relaxation rates increase due to CSA
Use i-HNCA instead of HN(CO)CA
03002-p.20