Introduction solution NMR Alexandre Bonvin Bijvoet Center for - - PowerPoint PPT Presentation

EMBO Global Exchange course, IHEP, Beijing

April 28 - May 5, 2011

Introduction solution NMR

Alexandre Bonvin

Bijvoet Center for Biomolecular Research

with thanks to Dr. Klaartje Houben

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NMR ‘journey’

• Why use NMR for structural biology...?
• The very basics
• Multidimensional NMR
• Resonance assignment
• Structural parameters
• NMR relaxation & dynamics

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Topics Why use NMR.... ?

NMR & Structural biology

Dynamic activation of an allosteric regulatory protein Tzeng S-R & Kalodimos CG Nature (2009)

apo-CAP CAP-cAMP2 CBD b c

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...allows to study the dynamics of biomolecular systems...

NMR & Structural biology

High-resolution multidimensional NMR spectroscopy of proteins in human cells Inomata K. et al Nature (2009)

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...structural studies in membrane and whole cells possible!

The Sample

• isotope labeling

– unlabeled (peptides) – 15N labelled (small proteins < 10 kDa) – 15N & 13C labelled (larger proteins, up to 30-40 kDa) – 15N, 13C & 2H labelled (large proteins > 40 kDa)

• protein production (E.coli)

– quite a lot & very pure & stable

• 500 uL of 0.5 mM solution -> ~ 5 mg per sample

– 13C labelling is costly, ~k! per sample – preferably low salt, low pH, no additives.

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• Pros...

– no need for crystal:

• no crystal packing artefacts, solution more native-like

– potential to study dynamics:

• picosecond to seconds time scales, conformational averaging,

chemical reactions, folding...

– easy study of protein-protein, protein-DNA, protein- ligand interactions

• Cons...

– NMR structure determination is a bit slow.... – Need isotope labeling (13C, 15N) – solution NMR works best for MW < 50 kDa

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Pros & cons of solution NMR in structural biology

The very basics of NMR

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precession E = µ B0

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Nuclear spin

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Nuclear spin

(rad . T-1 . s-1)

m = -! m = !

Larmor frequency

1H (I = 1/2)

Larmor frequency !H = "HB0 = 2#$H

13 14

Boltzman distribution

m = -! m = ! 1H (I = 1/2)

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Net magnetization

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Chemical shielding

Local magnetic field is influenced by electronic environment

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Chemical shielding

( )

! " # $ % = 1 2 B

Chemical shielding

• Chemical shift:

– δσiso [Hz] = νobs - ν0 – δσiso [ppm] = (νobs - ν0)/(ν0.10-6)

• ppm: parts per million
• ppm value is not field dependent

Chemical shift

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14 Tesla: !H = 600 MHz → 1 ppm = 600 Hz (1H) 21 Tesla: !H = 900 MHz → 1 ppm = 900 Hz (1H)

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Pulse

Observe with the Lamor frequency → “rotating frame”

FID: analogue vs digital

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Free Induction Decay (FID)

time (ms)

Signal

25 50 75 100 125 175 150 200

• freq. (s-1)

Signal

5 10 15 20 25 30 35 40

FT FT

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Fourier Transform

• NMR Relaxation

– Restoring Boltzmann equilibrium

• T2-relaxation

– disappearance of transverse (x,y) magnetization – 1/T2 ~ signal line-width

• T1-relaxation

– build-up of longitudinal (z) magnetization – determines how long you should wait for the next experiment

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Relaxation

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NMR spectral quality

• Sensitivity

– Signal to noise ratio (S/N)

• Sample concentration
• Field strength
• ..
• Resolution

– Peak separation

• Line-width (T2)
• Field strength
• ..

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Scalar coupling / J-coupling

H3C - CH2 - Br

3JHH

Multidimensional NMR

• multidimensional NMR experiments

– resolve overlapping signals

• enables assignment of all signals

– encode structural and/or dynamical information

• enables structure determination
• enables study of dynamics

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Why multidimensional NMR

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2D NMR

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3D NMR

1D

single FID of N points FID

t1

2D

N FIDs of N points

t2

FID

t1 mixing

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nD experiment

3D

NxN FIDs of N points

t2 t1 mixing t3 mixing

FID

direct dimension indirect dimensions

• mixing/magnetization transfer

spin-spin interactions

E = E =

???? proton A proton B

Encoding information

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• magnetic dipole interaction (NOE)

– Nuclear Overhauser Effect – through space – distance dependent (1/r6) – NOESY -> distance restraints

• J-coupling interaction

– through 3-4 bonds max. – chemical connectivities – assignment – also conformation dependent

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Magnetization transfer

t2

FID

t1

NOESY

tm

magnetic dipole interaction crosspeak intensity ~1/r6 up to 5 Å

COSY

t2

FID

t1

J-coupling interaction transfer over one J-coupling, i.e.

• max. 3-4 bonds

TOCSY

t2

FID

t1

J-coupling interaction transfer over several J-couplings, i.e. multiple steps over max. 3-4 bonds

mlev

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homonuclear NMR

t2

FID

t1

NOESY

tm A A (ωA) A B A (ωA) B (ωB) F1 F2 ωA ωA ωB

E = E =

proton A proton B ~Å

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homonuclear NMR

(F1,F2) = ωA, ωA (F1,F2) = ωA, ωB Diagonal Cross-peak

2D TOCSY

2D COSY & TOCSY

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HN Hα Hβ

2D COSY

HN Hα Hβ

– measure frequencies of different nuclei; e.g. 1H, 15N, 13C – no diagonal peaks – mixing not possible using NOE, only via J

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E = E =

1H 15N

heteronuclear NMR

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J coupling constants

1JCaCb = 35 Hz 1JCaC’ =

55 Hz

2JCaN = 7 Hz 1JNC’ =

• 15 Hz

1JCaN =

• 11 Hz

1JHN = -92 Hz 1JCaHa = 140 Hz 2JNC’ < 1 Hz 1JCbCg = 35 Hz 1JCbHb = 130 Hz

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J coupling constants

1JHN = -92 Hz

HSQC (heteronuclear single quantum coherence)

t2

FID

t1

1H

DEC

15N 1H 15N (ω15N) 1JNH 1JNH

(F1,F2) = ω15N, ω1H

J-mix block

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heteronuclear NMR

J-mix block

1H (ω1H)

1H-15N HSQC: ‘protein fingerprint’

39 note that spectrum is decoupled: no NH J- coupling

1H-15N HSQC: ‘protein fingerprint’

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J coupling constants

1JHN = -92 Hz 2JCaN = 7 Hz 1JCaN = -11 Hz

HNCA

t3

FID

t2

1H

DEC

15N

(F1,F2,F3)= (ω13Ca(i), ω15N(i), ω1H(i)) & (ω13Ca(i-1), ω15N(i), ω1H(i))

t1

13C 1H 15N 1JNH 1JNCa(i) 2JNCa(i-1)

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Triple resonance NMR

J-mix block J-mix block

15N (ω15N) 13C (ω13C) 1H (ω1H)

J-mix block

1JNCa(i) 2JNCa(i-1) 1JNH

J-mix block

Resonance assignment Structural parameters

a few months or more...

Structural study by NMR

• Sample preparation (months)
• Acquisition of NMR spectra (~1

month)

• Chemical shift assignments

– Backbone (days) – Side-chains (days)

• Analysis of NOESY spectra (weeks)
• Structure calculations (days)
• Functional studies with NMR

– Interaction with partner

RESTRAINTS

! dihedral angles ! distances between atoms ! orientation between bond vectors

Sources of structural information

– long-range NOEs – residual dipolar couplings – H / D exchange – effects of pH / T – effects of interacting partners – relaxation rates – .....

Secondary structure Tertiary structure

• OBSERVABLES

– chemical shifts (1H, 15N, 13C,

31P)

– J-couplings, e.g. J(HN,Hα)

φ ω ~ 180º N N C C C C C C O O φ ψ ω

RESTRAINTS: dihedral angles

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φ

anti-parallel β-strand α-helix

RESTRAINTS: dihedral angles

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φ ψ ψ φ

+180 ψ

• 180
• 180 φ +180

α-helix β-strand

Ramachandran plot

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φ -130 -60 ψ 125 -45 β-strand α-helix

• 13Cα and 13Cβ chemical shifts

– sensitive to dihedral angles – report on secondary structure elements

OBSERVABLE: chemical shift

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Karplus J = A.cos2(φ) + B.cos (φ) + C measured 3J(HNHα) reports on φ φ !

OBSERVABLE: homonuclear J- couplings

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φ !

RESTRAINT: distances

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• 1H-1H NOEs (2D NOESY, 3D NOESY-HSQC)

– signal intensity proportional to 1/r6 – reports on distance between protons

• distance restraints

OBSERVABLE: NOE

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Hx Hy

Cross-peak between Hx and Hy

• 1H-1H NOEs

– signal intensity proportional to 1/r6 – reports on distance between protons

• distance restraints

Sequential & medium range NOEs - SECONDARY STRUCTURE

Sequential

A B C D Z

• • • •

Intra-residue (used for identifying

spin-systems)

Medium range

OBSERVABLE: NOE

54 r = r =

NOEs in secondary structure elements

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A B C D Z

• • • •

Sequential Intra-residue (used for identifying

spin-systems)

Medium range

Long range NOEs - TERTIARY STRUCTURE

OBSERVABLE: NOE

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Longe range

• 1H-1H NOES

– signal intensity proportional to 1/r6 – reports on distance between protons

• distance restraints

• Tertiary information

– distances < 5 Å – Important structural information

Long-range NOEs

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anti-parallel

RESTRAINT: Orientation

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OBSERVABLE: Residual dipolar couplings

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No protein alignment ISOTROPIC SYSTEM D = 0

B0 Dij = " # i# j!µ0 4$ 2r3 3cos2 %(t) "1 2 Dipolar coupling Ω

OBSERVABLE: Residual dipolar couplings

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No protein alignment ISOTROPIC SYSTEM D = 0

B0 Dij = " # i# j!µ0 4$ 2r3 3cos2 %(t) "1 2 Dipolar coupling

Protein alignment ANISOTROPIC SYSTEM D ≠ 0

OBSERVABLE: Residual dipolar couplings

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JNH

ISOTROPIC

JNH + DNH JNH

ANISOTROPIC

⇒ Difference gives RDC DNH

B

• RDC reports on orientation of bond-vector

– orientation of bond-vector within an alignment tensor (defined by Aa and Ar) with respect to the magnetic field – i.e. orientation of bond vector with respect to other bonds

Residual dipolar coupling

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Long range orientational restraint - TERTIARY STRUCTURE

Dij = " # i# j!µ0 8$ 2r3 Aa(3cos2% "1) + 3 2 Ar sin2%cos(2&) ' ( ) * + ,

• Exchange 1H by D (2H)

– peaks disappear in time – accessibility of sites – stability of secondary structure elements

• H-bonds

OBSERVABLE: H/D exchange rates

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!1 !2 !3 increasing HN protection

N ! H""" O = C

kopen

# $ # #

kclose

% # # # N ! H

kint

# $ # N ! D

• Titration

– add in steps an interacting molecule (ligand / protein / DNA) – observe changes in chemical shift

• map interaction site

OBSERVABLE: chemical shift changes

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H1 7.0 8.0 9.0 N15 106.0 111.0 116.0 121.0 126.0 H1 7.0 8.0 9.0 N15 106.0 111.0 116.0 121.0 126.0

• Titration

– add in steps an interacting molecule (ligand / protein / DNA) – observe changes in chemical shift

• map interaction site

OBSERVABLE: chemical shift changes

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Key concepts structural parameters

• OBSERVABLES

– chemical shifts (1H, 15N, 13C, 31P) – J-couplings, e.g. 3J(HN,Hα) – medium-range NOEs – long-range NOEs – residual dipolar couplings (RDCs) – H / D exchange – effects of interacting partners – .....

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• RESTRAINTS

– dihedral angles – dihedral angles – medium range distances – long-range distances – orientations bond-vectors – accessibility / H-bonds – interaction surface – .....

Relaxation & dynamics

• ps

ns

s

ms s

RDC H/D exchange relaxation dispersion R1,R2,NOE

fs

bond vibrations

• verall tumbling enzyme catalysis; allosterics

loop motions domain motions side chain motions protein folding real time NMR J-couplings protein dynamics NMR

• 68

NMR time scales

• Fast motion

– Locally induced magnetic field changes – Causes relaxation

Binduced B0

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Local fluctuating magnetic fields

• Return to equilibrium

– Longitudinal relaxation → T1 relaxation

• Return to z-axis

– Transversal relaxation → T2 relaxation

• Dephasing of magnetization in the x/y

plane

Relaxation

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B0 B0

B1 B1

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Relaxation

• relaxation time is related to rate of motion

R1 = 1/T1 R2 = 1/T2

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FAST (ps-ns): rotation correlation time

• (b)

tail loop 1 3 N C

• residue number

5 10 15

• 157

177 197 217 237

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FAST (ps-ns): protein flexibility

R2/R1

• ps

ns

s

ms s

RDC H/D exchange relaxation dispersion R1,R2,NOE

fs

bond vibrations

• verall tumbling enzyme catalysis; allosterics

loop motions domain motions side chain motions protein folding real time NMR J-couplings protein dynamics NMR

• 74

NMR time scales

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SLOW (µs-ms): conformational exchange

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SLOW (µs-ms): conformational exchange

• Causes line-broadening

– Makes T2 relaxation faster

• wide range of time scales
• fluctuating magnetic fields
• rotational correlation time (ns)
• fast time scale flexibility (ps-ns)
• slow time scale (μs-ms): conformational

exchange

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Key concepts relaxation

The End

Thank you for your attention!