NMR Spectroscopy

Atom

Electron spins

Nucleus spins
Hydrogen atom

1945
Pauli
 Stein Gerlach Experiment at Uni Frankfurt: 1922

Otto Stern
Walther Gerlach

Carnegie-Mellon, Pittsburgh -> hired: Aksel Bothner-By

 NMR Spectroscopy

Atom

Electron cloud

nucleus spins
Hydrogen atom

1945 1944
Pauli Rabi
Magnetic Resonance
 NMR Spectroscopy

Atom

Electron cloud

Zavoisky

NMR: 1942: too low sensitivity

EPR: 1944

nucleus spins NMR: 1946

Hydrogen atom

Bloch Purcell

1945 1944 1952

Pauli Rabi
Bargon and Fischer, 1967, explained in 1969 by Kaptein: Radical pair mechanism
1200
2016
 Richard Ernst
Varian Associates

Weston Anderson
Varian Associates
 Fourier-Transformation (FT): 1804-1807
Jean Baptiste Joseph Fourier
Yesterday was my 21st birthday. At this
age Newton and Pascal had gained
immortality already.

FT
 Multi-D-NMR

Spin Physics
Nobelprice
in Chemistry
1991

Richard Ernst
ETH Zrich 1
H [ppm]

2.0

4.0

)
w ( 15N
11 10 9 8 7 6 5 4 3 2 1 0 ppm

w
1
(H
)
5.0 4.0 3.0 2.0 1.0
w (1H)
1
H [ppm]

1D 2D 3D
 3D NMR 1986

ETH
Utrecht
Selective labelling:
Arata, Tokyo
Oh & Markley, Ann Arbor

Non-Selective:
Kay, Klee, Bax
Gradients improve spectrum quality tremendously
 NMR based structural Biology

NMR Structure Determination:

Solution
Bacteria Molecular-
13C,15N-Source Sample Assignment
Biology
Multi-D-NMR

1) Residual Dipolar Couplings

J NH,CH
c

Nobelprice 2) N-H Relaxation Rates

In Chemistry

2002
J Coupling
HN
NOE/ROE~1/r
Cross Correlated Relaxation C'
(E.COSY, DQ/ZQ, FIDS, quant.J)
k+1

(NOESY, ROESY)
N

N
H k+1 R Ck
R

H
k
k
N
Hk+1 N
k+1
R R
H
k
Ck Nk+1
k+1
k N

R
Ck Nk+1 Ck Nk+1 C'
k
N

C' C' Ck Nk+1
N N

HN O
C'
N
HN
O HN O

Projection Restraints HN O
Distance Restraints Dihedral Restraints
1) Dipole-Dipole 1) Dipole-Susceptibility Tensor
2) Dipole-CSA 2) Dipole-Mass Tensor
Chemical Shift Restraints
3) CSA-CSA

Structure Calculation
(Simulated Annealing, Distance Geometry,
Kurt Wthrich Molecular Dynamics)

ETH Zrich &

Scripps Research
Institute, USA Bioinformatics 3D-Structure
 Residual Dipolar Coupling
Weak alignment through:
Lipid Bicelles
Magnetic Susceptibility
Liquid Crystalline Phase B0
Phage Particle Solution
Paramagnetic tags

H Dzz
B0 N
H 400
rMi rij
z N Dyy
x Dxx 40
y

ij
Dipolar coupling: Dres ~
rij3
[D ax(3cos -1)+
2 3
]
D sin2cos2
2 rh Prestegard
Tjandra & Bax
Extracting Residual Dipolar Couplings

Measurement of Coupling Constant without (reference)

and with lipid bicelles

Dcoupling = Danisotropic - Disotropic

Isotropic (reference) Anisotropic

115.7

115.7
116.1

116.1
[ppm]

[ppm]
116.5

116.5
93.38 Hz 96.97 Hz
116.9

116.9
15N

15N
117.3

117.3
117.7

117.7
7.66 7.65 7.64 7.63 7.62 7.61 7.66 7.65 7.64 7.63 7.62 7.61
1H [ppm] 1H [ppm]
Distinction between conformations

Xray model
Future

Prediction is very difficult, especially about the future: Niels Bohr

Improvement in hardware (higher fields, better probes)

and software (automation)

Increase in sensitivity: DNP, PHIP, CIDNP

Dynamics
Integration with other techniques:
Solid State NMR, EPR
Electron microscopy,
X-ray: diffraction, scattering (SAXS, WAXS)
Fluorescence
Molecular Dynamics
Qantum mechanics/Molecular Dynamics (QM/MM)

The battle is in the wetlab: intelligent labeling techniques

Become more physiological

 Dynamic Nuclear Polarization:
Becoming more sensitive
Polarization at 1.2 K, 3.35 T
Dissolution, liquid state NMR DNP in frozen state, MAS

Similar techniques: Xe totally polarized

Overhauser, by optical excitation of Rb
PRL 1953
CIDNP
para-Hydrogen induced polarization: PHIP, Altadena

C.P. Slichter

Phys.Golman et al. PNAS 2003

Rev. 1957 Griffin et al.
 Dynamic Nuclear Polarization
OH
O

Tumor OH B0
p p
OH

O
kT
electron 660 proton
O

e Signal enhancement
of nuclei

wave 1H,13C
1.1K
Dissolution and
observe in liquid
Prepolarizer state

Brindle, Nature Reviews 8, 94-107 (2008)

 NMR based structural
Biology

Dynamics
NMR Measurements for different
Time Scale of Motion Modulation of
Relaxation through chemical shift
Modulation of c
Dipolar coupling

dipolar couplings

ps ns s ms s
internal motions c secondary structural exchange Real time
element movement spectroscopy NMR
T1 T2 Domain
HetNOE movement
T2 T1 EXSY
 Binding entropy correlates with backbond order parameters
On the sub-nanosecond time scale: sub-c motion

Nature

dipolar couplings

ps ns s ms s
Relaxation c Relaxation dispersion exchange Real time NMR
dipolar couplings

ps ns s ms s
Relaxation c Relaxation dispersion
exchange Real time NMR
 Solution dynamics of ubiquitin:
Conformational selection instead of induced fit
: 30 X-ray structures
of ubiquitin with recognition proteins
o: Solution ensemble

900 ns

dipolar couplings
Lange and Lakomek et al. Science 2008

ps ns s ms s
Relaxation c Relaxation dispersion exchange Real time NMR
 Solution dynamics of ubiquitin:
The structures of ubiquitin in recognition protein
complexes are accessed by the EROS II ensemble

: 46 X-ray structures
of ubiquitin with recognition proteins
o: Solution ensemble
PCA modes taken from X-ray structures

O.F. Lange, N.A. Lakomek et al. Science 320, 1471-1475 (2008)

 Solution dynamics of TAR:
Conformational selection instead of induced fit:
RNA recognizing small molecules

19 ns
Al-Hashimi et al. Nature 2007

dipolar couplings

ps ns s ms s
Relaxation c Relaxation dispersion exchange Real time NMR
 Slow dynamics
Brutscher et al. PNAS 2007

dipolar couplings

ps ns s ms s
Relaxation c Relaxation dispersion exchange Real time NMR
Solid state NMR: Fibril structures

Meier et al. Science 2008

Focus on Methyl groups
Kay et al.
 Integration of Techniques: Example: NMR & EM
Others: NMR and X-ray, NMR and SAXS and SANS

Kessler et al. 1999

Combined liquid state NMR and X-ray
diffraction:VDAC 1
NMR Spectra, H/D Exchange, chemical shifts 2D Topology

Dihedral Angles Variation of Conformation Distance-Restraints

Se-Met Positions, Partial C-

Michael Habeck X-ray Amplitudes,
Chain Buster-TNT-Refinement experimental Phases

Bayrhuber et al. PNAS 105, 15370-5 (2008)

Combined liquid state & solid state NMR and
modelling and EM: PrgI
Stefan Becker, Adam
Lange et al. Nature 486,
276-279 (2012)
 -synuclein transformation from monomer to fibril: Toxicity of fibrils
and oligomers: Parkinsons disease

Solid state NMR

Fibrils
Oligomer
Monomer SQC-spindiffusion 50ms

Liquid state NMR

In vivo

In vitro

In vivo
Dimer
In vitro

TP
With TP we can T
decide what is toxic:
TP more toxic
A30P, than
A53T wt: Oligomers are toxic Lewy bodies
Cu(II)
TP less toxicpolyamines
than wt: Fibril are toxic
Partially folded Destabilized Unfolded -Synuclein
monomer monomer monomer
 100
C. elegans Days vs -syn wt)
Days vs -syn(TP)
100

% worms without neurite defects 90 80

Percent Survival
80

70 60
60

50
40
40

30
20
20

10
Drosophila Survival Curves
0
0
0 10 20 30 40 50 60
-Syn wild type TP Age in Days
Oligomers are toxic
Mouse neuron cells HEK Cells
100
% cells without defects

90
80 100%
70 TP
60
50 44%
40
In vivo
In vivo
30
20
10
0 homogeneous fibrils
-Syn wild type TP
EMBO J 28, 3256-3268 (2009) aggresome apoptosis
In cell NMR: becoming biological

Dtsch, Serber, Wagner, Selenko, Shirakawa

Interaction studies: becoming pharmaceutical

Fesik et al. Science 1996

Interaction studies: becoming pharmaceutical
Interaction studies: becoming pharmaceutical
Structure based Arg9 Tyr11
Binding model:

Leu13

Pro10 Ile12
Arg8

S. Luca et al., PNAS (2003) 100, 10706-10711

Correct Prediction of the biological activity of two ligands

4,4-spirolactam

Ile Leu

Arg Ile Leu

Arg Arg

Arg

~ 120 o ~ -120 o
(Pro) 146 15o
KD [nM] 0.27 12 16000

Bittermann, H., Einsiedel, J., Hubner, H. Gmeiner, P., 2004

Encounter complexes
 I wish that one of you will receive a
similar letter from Richard Ernst or Kurt
Wthrich!

Richard is becoming 80 this year! Kurt

becomes 75 this year!

So hurry up!
Good luck!