Introduction to

NMR spectroscopy
Swiss Institute of Bioinformatics
I.Phan & J. Kopp
NMR: the background

Complex technique. Requires knowledge in:

Mathematics

Phsics

Chemistr

!iolog

"Medicin#

In$ol$es a lot o% computing

N.M.R.

&uclear Magnetic Resonance

spectroscop

imaging

solid'state

....and much more

structure
imaging
dnamics
%olding
kinetics
in'$i$o
NMR applications
(inding
Can NMR be used for:

Medical diagnosis)

*rug'design)

Computing)
NUCLEAR Magnetic Resonance

&MR can detect atoms with a nuclear spin +,-

o!e nuclei "ith spin #$%
+..
/+
P
+..
+0
1
../2
+3
&
+.++
+/
C
00.04
+
5
&atural
a(undance
"6#
Isotope
12
C and
16
O do not have a spin

2
H,
14
N have spin 1
Is this good news %or
sol$ing the structure
o% proteins ( &MR)
NUCLEAR Magnetic Resonance

Can &MR (e used to detect:

78one)

9alt)

!utter)

: (rain tumor)
Nuclear MA&NE'IC Resonance

&uclei with spin ; (eha$e like a magnet

Placed in a constant magnetic %ield< the will

align with that %ield
H
5
!
5ow do we create a constant magnetic %ield)
NMR instru!entation

=he spectrometer
super'conducting
magnet
R1 %ield generator
sample tu(e
goes in here
&7=>:
the protein
is in solution
?+mM in water@
NMR is not sensiti(e

9ignals %rom the nuclei are measured in parts per

million ?ppm@ o% the static %ield strength

&MR experiments require:

concentrated samples

strong %ields A "$er# (ig magnets

)rotein structure file in the )rotein *ata +ank
9tructure data in the Protein *ata !ank
B C'ra crstallograph : o$er 4.6
B &MR : a(out +D6
'he cost of NMR

9uperconducting magnet : no resistance< no

current loss

Requires cooling to almost a(solute 8ero

Eiquid helium

"F Eiquid nitrogen %or insulation#

9trong magnetic %ield

Requires special in%rastructure

Minimi8e %ield distur(ance

&MR is a $er expensi$e techniqueG

superconducting magnet
radio %requenc unit
A typical NMR lab
Nuclear Magnetic RE,NANCE
5
5
5
5
5
5
5
5
R1 %ield
wa$e
to
%requenc
trans%ormation
sound
Nuclear Magnetic RE,NANCE
5
5
5
5
5
5
5
5
R1 %ield
wa$e
to
%requenc
trans%ormation
sound

In an &MR experiment< the energ input to make the nuclei

resonate is produced ( a Radio 1requenc "R1# PULSE
R1 %ield: magnetic %ield !
+
perpendicular to the constant
magnetic %ield !
.
time
current
in coil
t
pulse
I
Protein sample
:ppl and detect a
current in the coil
Constant magnetic %ield !
.
The )ULE
rotates the bulk magnetisation M
0

around !ais
The angle o" rotation
is proportional to the duration
o" the #$ pulse time t
!
.
-hat is the effect of the pulse.
NMR e/peri!ents

*epending on the length o% the pulses and dela

(etween pulses< di%%erent e%%ects are measured

Hariet o% &MR experiments: $ariet o% spectra

*epending on the %requenc o% the R1 pulse<

di%%erent nuclei can (e detected

Proton +5 I &MR

+3& I &MR spectra

+/C I &MR spectra

...etc...
0o" is an NMR signal detected.

:%ter the pulse< the nuclei return to their ground

energ state

=he nuclei precess (ack to their start position

Precessing induces a current that is detected ( a

coil in the &MR spectrometer

:s the nuclei return to equili(rium< the induced

current decreases (ack to 8ero A 1ree Induction
*eca "1I*#
time
I
'he NMR signal: a 1ree Induction
*ecay 21I*3
Current induced ( one precessing spin decas a%ter R1 pulse
=he 1I* signal
Nuclear Magnetic RE,NANCE

7ne spin: one (ar magnet

Man spins: (ulk magnetisation

*epending on the length o% the R1 pulse< the

(ulk magnetisation o% an ensem(le o% spins will
%lip at a di%%erent angle with respect to the static
%ield "!
.
#

:%ter the pulse< each spin precesses indi$iduall

and gi$es rise to an 1I*
)
1I* for an ense!ble of spins
Current induced ( precessing spins decas a%ter R1 pulse
Nuclear Magnetic RE,NANCE
5
5
5
5
5
5
5
5
R1 %ield
wa$e
to
%requenc
trans%ormation
sound
NMR signal processing

&MR spectrum: a superposition o% signals

7ne signal: 1I* o% one nucleus

Interpretation is made easier ( a simple

mathematical %ormula that trans%orms o% the 1I*
%rom the time domain to the %requenc domain

1ourier trans%ormation
I
1=
resonance
frequency
1=
1ourier 'ransfor!ation
frequency
time
time
Richard Ernst 2E'043

&o(el +00+
NMR structures

5ow do we get a protein /* structure with

&MR)

&MR experiments

*ata collection

9pectrum assignment

9tructure calculation

Method assessment: is &MR reall worth the e%%ort)

NMR e/peri!ents

+* &MR

Jhit< measureJ "+* pulse sequence#

R1
t
+
1=
#0 NMR e/peri!ent

+* &MR

+ peak %or each proton in a distinct en$ironment within

the protein

height num(er o% structurall identical 5 " 'C5/#

position "shi%t# electronegati$it o% surrounding

Minute di%%erences in shi%ts: measured in Part Per Million o% the %ield

width protein si8e

si8e expressed in terms o% tum(ling "correlation# time

'K there is an experimental limit to the si8e o% the proteins one can
determine ( &MR G we need tricks< more tricks....
1
H NM# spe%trum o" ethanol
>lectronegati$e group: proton signal is shi%ted compared to the re%erence
1
H NM# shi"ts in di""erent mole%ular environment
Lhat does it mean %or &MR spectra o%: lipids) 9ugars) *&:) Proteins)
#0 NMR spectra of proteins

Pro(lem &um(er +: o$erlap. =he larger the

protein< the more protons< the worse the o$erlap
9olutions:
' higher %ield< stronger magnet)
' -* experiments
' +3&< +/C la(elling
#0 NMR spectra of proteins

Pro(lem num(er -: in &MR terms< a large

protein ? K -3. residues@ means also (esides
o$erlapping peaks:

%aster decaing 1I*

(roader lines

poorer sensiti$it
&o practical solution %or this pro(lem ")#.
NMR e/peri!ents

-* &MR

Jhit< wait< hit< measureJ "-* pulse sequence#

R1
t
+
1=
R1
t
-
: slice %rom a(o$e gi$es
a map o% crosspeaks.
crosspeaks
The diagonal %ontains
the 1!dimensional spe%trum
O"" diagonal %ross peaks
%ontain ne" infor!ation
NMR e/peri!ents

Mse%ul -* &MR techniques

5'C79N: through'(ond connections $isi(le i% protons are at most /

(onds apart

5'&7>9N: through'space connections $isi(le if d < 6

C
5
C
5
C
5
C
5
peak on -* map with
position %"5#<%"5#
d
0eteronuclear e/peri!ents

Lhen o$erlap is too (ad %or sol$ing a structure

( +5 &MR alone

Ea(el protein with +3& and +/C

>xpensi$e< time'consuming< (ad ields

...(ut +3& and +/C resonate at completel di%%erent

%requencies %rom +5

Multi'dimensional experiments

... and much more...

0eteronuclear e/peri!ents
+3&'+5 C79N
15
N
1
H
&
5
C
5
NMR assign!ent

:ssignment o% spectra

method de$elopped ( K. Luethrich

%irst protein /*'structure sol$ed ( &MR in +04/

man protons< e$en in small proteins

complex pro(lem

relati$el simple solution

argua(l the most %astidious stage o% protein structure

determination ( &MR
5urt -uethrich 2E'043
&o(el -..-
NMR assign!ent

:ssignment o% spectra "K. Luethrich#

map indi$idual amino acids using C79N spectrum

set o% -* peaks particular %or each side'chain Jspin'sstemJ<

or relati$e arrangement o% protons
C,6 a!ino acid patterns
C
5
C
5
peak on -* map with
position %"5#<%"5#
*o ou see potential
pro(lems %or proteins)
*egeneracy of C,6 patterns
9ome amino acids share
the same spin sstem
... %or *&: itJs e$en worseG
NMR assign!ent

:ssignment o% spectra "K. Luethrich#

map indi$idual amino acids using C79N spectrum

set o% -* peaks particular %or each side'chain Jspin'sstemJ<

or relati$e arrangement o% protons

locate indi$idual amino acids within the sequence

using &7>9N spectrum "sequential assignment#

through'space connections %rom 5:"i# to 5&"iF+#

N,E6 se7uential assign!ent
&7>9N gi$es through'space connections:
peak on -* map with position %"5#<%"5#
i% distance d < 6
More fro! the N,E6 spectru!

9econdar structure patterns

Nuclear ,(erhauser Effect

=hrough'space connections< as gi$en ( the

&7>9N experiment< are the ke to sol$ing a
protein structure ( &MR

Eong'range interactions gi$e the %old o% the

protein chain
C
5
C
5
d
ad&a%ent beta!strands
'ositioning o" t(o heli%es
relative to ea%h other
NMR assign!ent

:ssignment o% spectra "K. Luethrich#

map indi$idual amino acids using C79N spectrum

set o% -* peaks particular %or each side'chain Jspin'sstemJ<

or relati$e arrangement o% protons

locate indi$idual amino acids within the sequence

using &7>9N spectrum "sequential assignment#

through'space connections %rom 5:"i# to 5&"iF+#

compile list o% all other peaks arising %rom through'

space connections "&7>s#

'K set o% pairwise distance restraints

NMR distance restraints
NMR structure calculation

5ow to get %rom the &7>s to the /* model)

pairwise distances
atomic coordinates
)
d
+
d
-
d
/
"x<<8#
"x<<8#
"x<<8#
NMR structure calculation

*istance geometr

sol$es the triangular inequalit

9imulated annealing "Michael &ilges#

%anc monte'carlo simulation "Otra$elling salesman

pro(lemO#

=orsion angle dnamics "Peter Pntert#

h(rid method in dihedral angle space

Michael Nilges 2EM+L3

CPE7R structure calculation and molecular

dnamics algorithm
)eter &uentert 2E'043

*N:&: structure calculation programme with

integrated automatic assignment protocol
NMR structures

Lhat ou see...
NMR structures

...is not what ou get %rom &MRG

NMR structures

&MR'deri$ed distance restraints "&7>s# are

upper'limits "Od Q D RO#

trans%ormation o% distances to coordinates gi$es man

solutions

&MR relies on cooperati$it o% distance restraints:

the more restraints per residue< the (etter de%ined the

structure

one &7> set produces a %amil o% structures:

loops: %ew experimental restraints 'K (ad de%inition 'K O%u88O

core: lots o% long'range restraints 'K good de%inition 'K OcompactO

NMR structure deter!ination

*i%%iculties

protein in solution: protein has to (e solu(le

insensiti$e method: requires high concentrations o% proteins

o$erlap: direct determination o% /* structures %or small proteins onl

"+3.'-.. residues#

:d$antages

no chemical modi%ication necessar

protein in solution: no crstal packing arte%acts< allows direct (inding

experiments< hdrodnamic and %olding studies

assignment o% la(ile regions possi(le: no gaps in structure

*etecting unstructured loops

&MR spectrum

spectrum shows no long'distance interactions (ut

sequential assignment is possi(le

(ack(one is %ree to adopt a range o% con%ormations:

greater $ariation in structure coordinates %or loop
residues

Crstallograph

electron densit map shows nothing at all

structures will ha$e gaps %or residues in mo(ile loops

)rion protein by NMR
&ot explora(le
( CRa
Riboso!e structure by 8Ray
Impossi(le task
%or &MR
NMR applications

9:R: Oshot'gunO approach to drug design

>xploring 1i(rils ( solid'state &MR

Protein %olding mechanism

&MR o% proteins in (icelles "semi'crstalline

state#

=R7>9N
tructure Acti(ity Relationship by NMR

*rug design

method de$eloped ( :((ott Ea(oratories

:im:

disco$er high'a%%init ligands %or proteins

>xample application:

anti$iral agent against the human papilloma$irus

9uantu! co!puting by NMR

Computers

molecules

In%ormation

atomic nuclei state

Programming

radio'%requenc pulse
9uantu! co!puting by NMR

%rom (its: . + to qu(its: S.<.K S.<+K S+<.K S+<+K

9uantu! co!puting by NMR

L5N)

atoms change energ states $er quickl

'' much more quickl than e$en the %astest computer processors.

each qu(it can take the place o% an entire processor

'' +<... ions o% (arium could take the place o% a +<... processor
computer.
9uantu! co!puting by NMR

L5:= sort o% pro(lem a quantum computer

would (e a(le to sol$e in principle)

Earge'scale crptograph

modelling and indexing $er large data(ases

9uantu! co!puting by NMR

L57 wants to (uild a quantum computer)

I!M,MI=,!erkele, 9tan%ord

Isaac Chuang "I!M# &eil Pershen%eld "MI=#

T>na(ling technologU %or &MR'(ased quantum computingV scale up

to +.'W. qu(its

5ar$ard, MI=,Eos :lamos

*a$id Cor "5ar$ard#

Xuantum algorithms and &MR'(ased sstems

7x%ord Mni$ersit

*a$id *eutsch< Jonathan Jones

Ion'trap and &MR implementationsV quantum in%ormation theor

finally...the R.Ernst (ie" of
NMR
mathematics
phsics
chemistr
(iolog
medicin
&
M
R