3D Modern Techniques

EPJ Web of Conferences 30, 03002 (2012) DOI: 10.1051/epjconf/20123003002

© Owned by the authors, published by EDP Sciences, 2012

Organized by

Thibault Charpentier [email protected]

Patrick Berthault

[email protected]

Constantin Meis

[email protected]

Experiment and Modelling in Structural NMR

November 28th – December 2

nd

₂₀₁₁

INSTN – CEA Saclay, France

Nicolas Birlirakis

ICSN CNRS, France

3D modern techniques

“Experiment and Modelling in Structural NMR”

ORGANISATION • 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 Denis MERLET, University Paris-Sud 11

• 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]

November 28 - December 2, 2011 INSTN - CEA, Saclay http://www-instn.cea.fr (events 2011)

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 Train-ing in the afternoon involvTrain-ing both the theoretical and experi-mental aspects.

A limited number of scholarships can be obtained for PhD students

contact : [email protected]

Lectures

History, General principles M. Goldman, CEA Saclay Formalism and Interactions H. Desvaux, CEA Saclay Molecular Modelling M. Nilges, Institut Pasteur

Anisotropy for

Conforma-tional 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

Interactions

ligand-macromolecules G. Lippens, University of Lille

Bio-Solid State NMR

Bioinformatics-protein assembly

H. Oschkinat, University of Berlin

R. Guérois, CEA Saclay

Materials Modelling &

Simulations C. Meis, INSTN - CEA, Saclay DFT calculations of NMR

parameters. J. Yates, Oxford University Inorganic Materials-

Ceramics S. AshbrookSt Andrews , University of 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 Sys-tems over many decades

R. Kimmich, University of Ulm

 

Winter-School



3D Modern Techniques N. Birlirakis ICSN - CNRS

3D NMR experiments applied to proteins

H

C COO

-+_H3N

R

*

amino acid 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

+_H 3N C

H

R1

C

O

N H

C H

R2 C

O

N C

H H

-[

NMR studies of peptides (<50aa)

General considerations

- Chemically synthesized molecules (SPPS)

- Isotopic labelling expensive → homonuclear 1_H-1_{H 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 from3_{JHN-Hα couplings: 2D-COSY-DQF (Karplus equations)}

- Interproton distance determination: 2D NOESY (build up with several τ_m)

H

C C N

CH3

N C

H

CH C O

H3C CH3

H

O H

P E M A

t1

t1 1/4J 1/4J

]

n

t1

COSY

RELAY

TOCSY

NOESY t1 τm _ROESY t1 SL

antiphase transfer

in-phase transfer

CSL

NMR studies of small proteins (50-100aa)

General considerations

- Favorable T2 relaxation

- Increased 1_{H 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)

Breakthrough

- Recombinant protein expression → affordable 15_{N labelling (natural abundance 0.366%)}

P E1 M1 E2 M2 A

t1 τm t2

- 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 15_NH4Cl

Heteronuclear 3D experiments

- Resolve the 1_{HN overlapping by the use of} 15_{NH frequency : 4ppm versus 30-35ppm}

Sensitivity considerations (S/N)

S ~ n A (1/T) Bo3/2γobs3/2γexc T2 √Nscan Cryoprobe technology: N↓

NMR studies of small proteins (50-100aa)

n: sample quantity (v*c) A: active nuclei abundance

t1

[

]

τm

1/4J 1/4J 1/4J 1/4J

1_H

15_N t2 cpd

t3

NOESY(TOCSY) - fHSQC

φ₁

φ₂

Gr

1_{Hj /} 1_Hi

15_NH i

1_HNi

WG

TOCSY mixing: use clean CSL schemes to suppress dipolar cross relaxation and NOESY/ ROESY artifacts

y

Going from 2D to 3D

ppm

10 8 6 4 2 0 ppm

10 8 6 4 2 0

F3 [ppm]

10 9 8 7

F2[p pm]

13

0

125 120 115 110

F1

[p

pm

]

8 6

3D NOESY-HSQC H-H-N (τ_m=150ms)

8 scans

2D NOESY (τ_m=150ms)

3D data analysis

9.0 8.5 8.0 7.5 7.0

8 6 4 2

9.0 8.5 8.0 7.5 7.0

ω3 - 1_{H (ppm)} L115H-E113N-H

M109HA-E113N-H

G110H-E113N-H A112HA-E113N-H

A112QB-E113N-H

E113HG3-N-H A116QB-E113N-H

G110HA2-E113N-H G110HA3-E113N-H E113HA-N-H

E113HG2-N-H E113HB#-N-H

F114H-E113N-H

A112H-E113N-H ?

9.0 8.5 8.0 7.5 7.0

8 6 4 2

? ?

clean TOCSY-HSQC (tm=70ms) NOESY-FHSQC (tm=150ms) or ROESY-HSQC([email protected]°)

ω

1 -

1H (ppm)

Heteronuclear correlation building blocks

1/2J 1/2J

1_H

15_N t1 cpd

t2

HMQC (JII, JSS, T2I, T2S)

φ₁

1/4J 1/4J 1/4J 1/4J

1_H

15_N t1 cpd

t2

φ₁

HSQC (JSS, T1I, T2S) y

-Iy 2Ix,ySz

-2Ix,ySy

2Ix,ySz Iy

-Iy 2IxSz

-2IzSy

2IySz

2IySxS -I2IySxx

refocused HSQC (JSS, T2S)

1/4J 1/4J 1/4J 1/4J

1_H

15_N t1 cpd

t2

y

-Iy 2IxSz

-2IzSy

2IxSz Iy

1/4J 1/4J

φ₁

cpd

Sx

1/4J 1/4J

-2IzSy

-2IzSx

Axial peak suppression: φ₁ and rec cycled x, -x

Quadrature detection at the indect dimension using φ₁ incrementation:

Heteronuclear correlation building blocks

1/2J 1/2J

1_H

15_N t1 cpd

t2

HMQC (JII, JSS, T2I, T2S)

φ₁

1/4J 1/4J 1/4J 1/4J

1_H

15_N t1 cpd

t2

φ₁

HSQC (JSS, T1I, T2S) y

-Iy 2Ix,ySz

-2Ix,ySy

2Ix,ySz Iy

-Iy 2IxSz

-2IzSy

2IySz

2IySxS -I2IySxx

refocused HSQC (JSS, T2S)

1/4J 1/4J 1/4J 1/4J

1_H

15_N t1 cpd

t2

y

-Iy 2IxSz

-2IzSy

2IxSz Iy

1/4J 1/4J

φ₁

cpd

Sx

1/4J 1/4J

-2IzSy

-2IzSx

1/4J 1/4J 1/4J 1/4J

1_H

15_N t1 cpd

t2

φ₁

sensitivity enhanced HSQC (JSS, T1I, T2S) y

-Iy 2IxSz

-2IzSy 2IySz 2IySx

-Ix 2IySx -2IzSy

-2IzSx

1/4J 1/4J

±y

Iz ±2IySz

y

-Iz ±2IySz

Iy ± Ix

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 (3_J

NMR studies of medium size proteins (100-200aa)

- Recombinant expression of 13_C, 15_{N labelled proteins using} 15_NH

4Cl and 13C6-glucose

→ access to triple-resonance experiments

- Resolve the 1_{HC overlapping by using} 13_{CH frequency: 5ppm versus 65-70ppm}

for the aliphatics

- Use of heteronuclear couplings 1_H-15_N, 1_H-13_C, 13_C-13_C, 15_N-13_{C (}1_{J and} 2_{J) for}

unambiguous intra- and inter-residual assignment

- Magnetisation transfers implicating slowly relaxing nuclei

i i-1 N Cα Hα Cβ C O N H Cα Hα Cβ C O H 140Hz 55Hz 55Hz

90Hz 90Hz 140Hz

35Hz _{7Hz <1Hz}

15Hz 11Hz

Backbone assignment via

out and back

3D experiments

HNCO (100)

N Cα H Hα

C O N H Cα Hα C i i-1 O

HNCA (50/15)

N Cα

H Hα

C O N H Cα Hα C i i-1 O

HN(CA)CO (13/4)

N Cα

H Hα

C O N H Cα Hα C i i-1 O

HN(CO)CA (71)

N Cα

H Hα

C O N H Cα Hα C i i-1 O

HNCACB (4-1.7/1.3/0.5)

N Cα

H Hα

C O N H Cα Hα C i i-1 O Cβ Cβ

HN(CO)CACB (13-9)

N Cα

H Hα

C O N H Cα Hα C i i-1 O

The HNCA experiment

∆1 ∆1 1_{H (I)}

15_{N (S)}

13_Cα(T)

13_CO

y

-Iy 2IxSz

-2IzSy

2∆1

∆2 ∆2

2SyTz

t2/2 φ2 -2SzTy WALTZ-y -Ix Gz I:0 I:+1 I:-1 S:0 S:+1 S:-1 S:0 S:+1 S:-1 S:0 S:+1 S:-1 T:+1 T:-1 T:0 T:+1 T:-1 T:0 T:+1 T:-1 T:0 T:+1 T:-1 T:0 T:+1 T:-1 T:0 T:+1 T:-1 T:0 T:+1 T:-1 T:0 T:+1 T:-1 T:0 T:+1 T:-1 T:0 2SyTz

∆2−t1/2

2∆1

-2IzSy

∆1 ∆1

φ1

∆2

CPD t1/2

BS

WG

BS

t2/2

2IySz y y y y -x

The HNCA experiment

∆1 ∆1 1_H

15_N

13_Cα

13_CO

y 2∆1

∆2 ∆2

t2/2

WALTZ-y

Gz

∆2−t1/2

2∆1 ∆1 ∆1

∆2

CPD t1/2

BS

WG

BS

t2/2 y

y

-x

Water magnetization at z prior to WATERGATE module

Proton decoupled version (longer T2 relaxation for 15N, 13C nuclei)

Constant time at 15_{N dimension} 1_J

CαCβ couplings not refocused during t2

∆1 = 1/41J_HN = 2.7m

∆2 = 12m (relaxation optimized, 1/4J_NCα ~ 22m)

Same sequence for HNCO experiment (via Cα-CO channel interconversion) HNi

NHi

13_Cα i,i-1

15_NH i

1_HN i

y φ1 y

Experimental aspects of triple

resonance experiments

- Shaped pulses

- Pulsed field gradients

- Water suppression

Hard pulses

Excitation profile of a rectangular 90° pulse τp = 1ms

t

τp

B

Rectangular hard pulses (polychromatic): γB1/2π >>> ∆F

Typical hard pulse lengths @ 800MHz, max admitted power:

1_{H → 7-...µs} 15_{N → 39.5µs}

sinc (=sinx/x)

15000 10000 5000 0 -5000 -10000 -15000 _Hz

75 50 25 0 -25 -50 -75 ppm

0 600 -600

(DRX-800, 13_C)

1/τp

Z

X

Y

θ Flip angle θ

depends on γB1τp

Shaped selective pulses

3000 2000 1000 0 -1000 -2000 -3000 Hz

[points] 200 400 600 800

Amplitude

Phase

[points] 200 400 600 800

Rectangle

90°

3000 2000 1000 0 -1000 -2000 -3000 Hz

Triangle

90°

3000 2000 1000 0 -1000 -2000 -3000 Hz

90° Sinc

[points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

[points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

Amplitude modulation: first trials

Shaped selective pulses

Amplitude modulation: peak selection

Gaussian_1

90° 270°

3000 2000 1000 0 -1000 -2000 -3000 Hz [points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

Gaussian_1

[points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

3000 2000 1000 0 -1000 -2000 -3000 Hz

180°

3000 2000 1000 0 -1000 -2000 -3000 Hz

Gaussian_1

[points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

90° Gaussian δ and J evolution during pulse:

270°

τ

90°x

180°y, -y

Shaped selective pulses

Amplitude/phase modulation: band selection (top-hat profile)

Gaussian Cascade and BURP famillies

90° 180° 90°

U-BURP 2

6000 4000 2000 0 -2000 -4000 -6000 Hz 6000 4000 2000 0 -2000 -4000 -6000 Hz 6000 4000 2000 0 -2000 -4000 -6000 Hz

Cascade Q5

[points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

Cascade Q3

[points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

[points] 200 400 600 800 [points] 200 400 600 800

Amplitude

Phase

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

=

τ

∆Ω

Examples of selective excitation

1D spectrum of strychnine in CDCl3

Advantages

• 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

•

• •

• •

2.0 3.0 4.0 5.0 6.0 7.0

8.0 ppm

- of one peak - of a spectral region

- of two peaks - of several peaks

Examples of 2D selective experiments

F2

F1 t1 tm

90° 90° 90°

t1 tm 90°

90° 90°

t1 tm 90°

90°

90°

tm 90°

90° 90°

classical NOESY

semi-soft NOESY

soft NOESY

1D selective NOESY

t2

t2

t2

Selective excitation in triple resonance experiments

- Differentiation between 13_{Cα (or} 13_{Caliph) and} 13_{CO regions}

Use of rectangular pulses:

τ

τ

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 13_{C and} 15_{N pulses}

Use of universal top-hat pulses (or interleaved G4 and G4tr)

- Selective perturbation of 1_{HN region (BEST experiments)}

- Solvent selection (flipback or suppression)

-0.80 -0.60 -0.40 -0.20 -0.00 0.20 0.40 0.60 0.80

30000 20000 10000 0 -10000 -20000 -30000 Hz

180° pulse comparison: 33µs square and 200µs Q3

CO Caliph

-0.80 -0.60 -0.40 -0.20 -0.00 0.20 0.40 0.60 0.80

30000 20000 10000 0 -10000 -20000 -30000 Hz

90° pulse comparison: 36µs square and 300µs G4

CO Caliph

ATTENTION

Offset-dependent

transient Bloch-Siegert phase shifts in

homorefocusing

Broadband inversion and decoupling

- High field spectrometrs → large13C spectral width in Hz → imperfect inversion/decoupling

→ use of adiabatic pulses: smoothed CHIRP and WURST families

Adiabatic decoupling:

Use P4M5 supercycle

Use bi-level decoupling for reduction

of sidebands in 1_H-1_H-13_{C and} 1_H-1_H-15_N

experiments

Homonuclear decoupling → offset

dependent Bloch-Siegert frequency shifts

- Adiabatic 15_{N 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

-0.80 -0.60 -0.40 -0.20 -0.00 0.20 0.40 0.60 0.80

30000 20000 10000 0 -10000 -20000 -30000 Hz

180° pulse comparison: 28µs square and 500µs CA-WURST-4

Pulsed field gradients

Linear variation of B0 at a certain direction

- Coherence transfer pathway selection:

Σ γ

- Artifact suppression per scan ie 1_H12_{C artifacts with short phase cycling (...substraction)}

- Solvent suppression and control of water magnetisation (radiation damping) - Translational diffusion measurements

- Magnetic Resonance Imaging

1/4J 1/4J 1/4J 1/4J

1_H

15_N t1 cpd

t2

φ₁ y

1/4J 1/4J

±y y

80 ±8

gradient sensitivity enhanced HSQC (JSS, T1I, T2S)

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

1/4J 1/4J

1_H

15_N

φ₁

y

-Iy 2IxSz 2IzSz

SLx

Iy -Iy

Gr

1_H

Gr

90°y-y 90°y

The HN(CA)CO experiment

∆1 ∆1

1_H

15_N

13_Cα

13_CO

y 2∆1

∆2 ∆2

t2/2 φ₂

WALTZ-y

Gz

∆2−t1/2

2∆1 ∆1 ∆1

∆2

CPD t1/2

BS

WG

BS

t2/2 y

y

-x

INEPT version ∆1 = 1/41J_HN = 2.7m

∆2 = 12m (relaxation optimized, 1/4J_NCα ~ 22m)

∆3 = 3.5-4m (relaxation optimized, 1/4J_COCα = 4.5m)

HNi

NHi

13_Cα i,i-1

15_NH i

1_HN i

y φ1 y

BS y

y

∆3 ∆3 ∆3 ∆3

13_CO i,i-1

The HN(CO)CA experiment

∆1 ∆1 1_H

15_N

13_CO

y 2∆1

∆2 ∆2

t2/2 φ2

WALTZ-y

Gz

∆2−t1/2

2∆1 ∆1 ∆1

∆2

CPD t1/2

WG

BS

t2/2

y -x

mixed INEPT / DQ version HNi

NHi

13_Cα i,i-1 (DQ)

15_NH i

1_HN i

y φ1 y

BS

y

∆3 ∆3

13_CO i,i-1

13_Cα

The HNCACB experiment

∆1 ∆1

1_H

15_N

13_Cα

13_CO

y 2∆1

∆2 ∆2

t2/2

WALTZ-y

Gz

∆2−t1/2

2∆1 ∆1 ∆1

∆2

CPD t1/2

BS

WG

BS

t2/2 y

y

-x

mixed INEPT / RELAY sequence ∆1 = 1/41J_HN = 2.7m

∆2 = 12m (relaxation optimized, 1/4J_NCα ~ 22m)

∆3 = 3.6m (relaxation optimized, 1/4J_CαCβ = 7.1m) 1_J

CαCβ and 1JCβCγ couplings not refocused during t2

Cα, Cβ peaks of opposite sign

HNi

NHi

13_Cα i,i-1

15_NH i

1_HN i

y φ1 y

φ1 φ3 y

13_Cα i,i-1 13_Cα,β

i,i-1

∆3 ∆3 ∆3 ∆3

Rapid pulsing - BEST experiments

1/2J 1/2J

1_H

15_N t1 cpd

t2

SOFAST-HMQC (JSS, T2I, T2S)

φ₁

1/4J 1/4J

1_H

15_N t1 cpd

t2

1

BEST-HSQC (JSS, T1I, T2S)

y

φ

1/4J 1/4J

: PC9 pulse (flip-angle 120°, Ernst angle) band-selective excitation pulse

: band-selective universal 180° pulse

: PC9 pulse (flip-angle 90°) band-selective 90° pulse

: band-selective universal 180° pulse

- In 1_H-15_{N correlation experiments} 1_{HN longitudinal dipolar relaxation is accelerated} if the rest of the protein protons (aliphatics) and 1_H

Rapid pulsing - BEST experiments

7 8

9

10 ppm

105

110

115

120

125

130

7 8

9

10 ppm

105

110

115

120

125

130

SOFAST-HMQC BEST-HSQC

90° pulse → 1770µs Q5.1000

1_{H carrier frequency @ 7.999ppm}

180° pulse → 1260µs Q3.1000

P5M4 15_{N 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

∆1 ∆1

1_H

15_N

13_Cα

13_CO

y

2∆1

∆2 ∆2

t2/2

2

Gz

∆2−t1/2

φ1

CPD ∆2

t1/2

BS BS

t2/2

y y

φ

∆1 ∆1

y

y

y

: 180° broadband inversion pulse → 155µs power-BIBOP pulse

3D spectra for some BEST

out and back

experiments

F3[ppm]

10 9 8 7

F2[p pm ] 13 0 12 5 120 11 5 11 0 F1 [p pm ] 65 60 55

BEST-HNCA: 4scans, 1h 53min

F3[ppm]

10 9 8 7

F2[p pm ] 13 0 12 5 120 11 5 11 0 F1 [pp m] 18 0

BEST-HN(CO)CA: 4scans, 2h

BEST-HNCO: 4scans, 58min

F3[ppm]

9 8 7

F2 [ppm ] 13 0 125 12 0 115 11 0 F1 [pp m] 17 6 17 4 10 F3[ppm]

10 9 8 7

F2 [ppm] 130 12 5 12 0 115 11 0 F1 [pp m] 58 56 54

BEST-HN(CA)CO: 4scans, 59min

F3[ppm]

10 9 8 7

F2[pp m] 13 0 12 5 120 11 5 110 F1 [pp m] 60 50 40

BEST-HNCACB: 4scans, 3h 54min

F3[ppm]

10 9 8 7

F2[pp m] 13 0 12 5 120 11 5 11 0 F1 [pp m] 19 0 17 0

BEST-HN(CO)CACB: 4scans, 4h 9min

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

Backbone and side chain assignment

via

transfer

3D experiments

HACACO

N Cα H Hα

C O N H Cα Hα C i i-1 O

N Cα H Hα

C N H Cα Hα C Cβ Cγ Cδ Hβ Hγ Hδ O

H(C)CH-TOCSY / (H)CCH-TOCSY

O

i i-1

N Cα H Hα

C N H Cα Hα C O

(H)CC(CO)NH-TOCSY / H(CCCO)NH-TOCSY

O i i-1 Cβ Cγ Cδ Hδ Hγ Hβ

N Cα H Hα

C N H Cα Hα C Cβ Cγ Cδ Hβ Hγ Hδ O HACACO-TOCSY O i i-1

N Cα H Hα

C N H Cα Hα C Cβ Hβ O HBHA(CBCACO)NH i i-1 O In H2O:

In D2O:

The HCCH-TOCSY experiment

∆1

CPD

BS

∆1

t1/2 t1/2

∆1/2 ∆1/2 y

t2/2 t2/2 _FLOPSI

y -y

∆1/2 ∆1/2

∆1 1_H 13_C 13_CO Gr purging

Refocused INEPT / z-TOCSY

Constant time at both indirect dimensions

∆1 = 1/21J_HC = 37m (optimized for CH₂)

Purging of residual water magnetization with B0 and RF gradients

∆1

NMR studies of large proteins (>200aa)

- Decrease transverse 13_{CH 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 15_N1_{H 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)

- At high fields transverse 13_{CO relaxation rates increase due to CSA}

Use i-HNCA instead of HN(CO)CA

15_N

1_H

Non-decoupled HSQC