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 permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 (> ∆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
