Protein structure determination by NMR

?
Brian J Goodfellow!
Departamento de Quimica!
Universidade de Aveiro!
Aveiro 3810-193!
Email: brian.goodfellow@ua.pt
NMR

NMR - Bibliography

NMR

NMR - Bibliography
- Teng, Q. Structural Biology: Practical NMR applications Springer
Science, USA (2005)
- Evans, J.N.S. Biomolecular NMR Spectroscopy, Oxford University
Press (1995)
- Wthrich, K. NMR of Protein and Nucleic Acids, Wiley-Interscience
Pub., (1986)
- Levitt, M.H. Spin Dynamics. Basics of Nuclear Magnetic Resonance,
John Wiley & Sons, Ltd, England (2002)
- Gil, V.M.S., Geraldes, C.F.G.C. Ressonncia Magntica Nuclear.
Fundamentos e aplicaes, Fundao Calouste Gulbenkian, ed. (1988)
- Claridge, Timothy D. W. High Resolution NMR Techniques in Organic
Chemistry, Tetrahedron Organic Chemistry Series, Vol 27, Elsevier, 2nd
Ed (2009)
- Friebolin, H. Basic One- and Two-Dimensional NMR Spectroscopy,
VCH publishers, New York-Germany, 2a ed. (1993)
NMR

NMR - Nobel Prizes

Nobel prize - Physics 1952

Felix Bloch
Stanford,USA

NMR

Discovery of the NMR effect

Edward Mills Purcell,

Harvard, USA

NMR - Nobel Prizes

Nobel prize - Chemistry 1991

FT-NMR, 2D NMR

Richard R. Ernst - ETH

Nobel prize - Chemistry 2002

Protein Structure determination by NMR and MS

J.B. Fenn - USA, K. Tanaka - Japan, K. Wthrich - ETH

NMR

NMR - Nobel Prizes

Nobel prize - Medicine 2003

NMR Imaging

Lauterbur - USA

NMR

Mansfield - UK

Principais aplicaes RMN!

!
Elucidao estrutural!
!

Produtos naturais!

!
!

Qumica orgnica. Ferramenta analtica de eleio dos qumicos de

sntese!

Estudo de processos dinmicos!

!

Cintica de reaces!

Estudo de equilbrio (qumico ou estrutural)!

Estudos estruturais (tridimensionais)!

!

protenas DNA/RNA. Complexos de protenas com DNA/RNA!

Drug design - structure activity relationship (SAR) por RMN!

Medicina - Magnetic Resonance Imaging (MRI, fMRI)
NMR

NMR

DQ tem maior numero de espctrmetros RMN no pas - 5

400MHz - solid state

300MHz - service

probe
500MHz - metabolomics, LCNMR

500MHz - natural products

(cryo probe)

700MHz - solids, proteins

E Cabrito - FCTUNL

O espectro de RMN
Desvio qumico (!), constante de acoplamento (J), largura
de linha e intensidade
Espectro de proto do etanol
-CH2-

CH3

-OH + H2O

campo baixo

desblindado

campo alto

1H,

blindado

NMR

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Fundamentos
RMN - Detecta a absoro de radiofrequncias (radiao
electromagntica) por certos ncleos numa molcula
Para descrever o fenmeno na totalidade necessria
alguma (muita mesmo) mecnica quntica

SPIN
Ao contrrio da massa atmica e da carga o spin no tem
equivalente macroscpico, simplesmente existe...

NMR

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Fundamentos
S os ncleos com nmero quntico de spin (I) ! 0
podem absorver/emitir radiao electromagntica
massa atmica e nmero atmico par, I = 0
(12C, 16O)
I = 0

Ver tabelas para

tirar I

massa atmica par e nmero atmico impar, I = inteiro

(14N, 2H, 10B)
I = n inteiro
massa atmica impar, I = meio-inteiro
I = 1/2
(1H, 13C, 15N, 31P)

Os estados de spin de um ncleo (m) esto quantizados:

m = I , (I - 1), (I - 2), ..., - I

NMR

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Fundamentos
Para 1H, 13C, 15N, 31P

m = 1/2, - 1/2
estes ncleos s podem existir em dois estados de spin

Momento magntico nuclear ():

L!

NMR

="Ih/ 2 #

" razo magnetogrica

h constante de Plank

um vector que d a direco e a magnitude do

magneto nuclear

B Volkman - MCW

Effect of a magnetic field (for I = 1/2)!

!

In the ground state (no magnetic field) all nuclear spins are disordered, and
there is no energy difference between them. They are degenerate!

!
!
!
!
!
!
!

= h / 4

!
!
!
!
!
!
!
!

Bo

Since they have a magnetic moment, when we apply a strong!

external magnetic field (Bo), they orient either against or with it:!

There is always a small excess of nuclei (population excess)!

aligned with the field than pointing against it.

NMR

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Energia e populaes
Quanto maior B0, maior a diferena de energia

"E = " h B0 / 2 #
E

$%

%
B0

$
B0
NMR

&E

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Energia e populaes
Quanto maior B0 maior a diferena de energia.
A razo das populaes dos dois estados depende de "E e
pode ser calculada atravs da distribuio de Boltzmman

N$ /N% = e"E/kT
A "E para 1H a 400 MHz (B0 = 9.4 T) 3.8 " 10-5 kcal/mol
N$ /N% = 1.000064
num milho de spins s h uma diferena de 64
A RMN uma tcnica muito pouco sensvel (pelo menos quando
comparada com IV ou UV)

NMR

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Energia e sensibilidade
Ncleos com " elevado iro absorver/emitir mais energia e
como tal sero mais sensveis.
A sensibilidade proporcional a N$ /N% e ao fluxo magntico
da bobine e ambos dependem de ".
No total a sensibilidade depende de "3
" 13C = 6,728 rad / G
" 1H = 26,753 rad / G

1H

# 64 vezes mais sensvel que 13C s

devido a "

se se considerar tambm a abundncia natural do 13C (# 1%) verifica-se que

13C 6400 vezes menos sensvel que 1H

NMR

Useful nuclei such as 15N, 13C are rare

Isotope Spin
(I)

1H
2H
13C
14N
15N
17O
19F
23Na
31P
113Cd

1/2
1
1/2
1
1/2
5/2
1/2
3/2
1/2
1/2

Natural

Magnetogyric ratio

abundance

g/107 rad T-1s-1

99.985 %
0.015
1.108
99.63
0.37
0.037
100
100
100
12.26

26.7519
4.1066
6.7283
1.9338
-2.712
-3.6279
25.181
7.08013
10.841
-5.9550

NMR frequency
MHz (2.3 T magnet)

100.000000
15.351
25.145
7.228
10.136783
13.561
94.094003
26.466
40.480737
22.193173

NMR

Useful nuclei such as 15N, 13C are rare

Isotope Spin
(I)

1H
2H
13C
14N
15N
17O
19F
23Na
31P
113Cd

NMR

1/2
1
1/2
1
1/2
5/2
1/2
3/2
1/2
1/2

Natural

Magnetogyric ratio

abundance

g/107 rad T-1s-1

99.985 %
0.015
1.108
99.63
0.37
0.037
100
100
100
12.26

26.7519
4.1066
6.7283
1.9338
-2.712
-3.6279
25.181
7.08013
10.841
-5.9550

NMR frequency
MHz (2.3 T magnet)

100.000000
15.351
25.145
7.228
10.136783
13.561
94.094003
26.466
40.480737
22.193173

Useful nuclei such as 15N, 13C are rare

Isotope Spin Natural
(I)
abundance

1H
2H
13C
14N
15N
17O
19F
23Na
31P
113Cd

1/2
1
1/2
1
1/2
5/2
1/2
3/2
1/2
1/2

99.985 %
0.015
1.108
99.63
0.37
0.037
100
100
100
12.26

Magnetogyric ratio
(/107) rad T-1s-1

NMR frequency
MHz (2.3 T magnet)

26.7519
4.1066
6.7283
1.9338
-2.712
-3.6279
25.181
7.08013
10.841
-5.9550

100.000000
15.351
25.145
7.228
10.136783
13.561
94.094003
26.466
40.480737
22.193173

NMR

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Energia e populaes
A energia de um spin num campo magntico vai depender
!
do campo magntico, B0, e do momento magntico

E = - . B0

E$ = - " h B0 / 4 #

momento magntico alinhado com o campo

magntico, B0

"E = " h B0 / 2 #

B0

E% = " h B0 / 4 #

momento magntico alinhado contra o campo

magntico, B0

NMR

B0

Energia e frequncia
A energia est relacionada com a frequncia...

"E = h '0

'0 = " B0 / 2 #

"E = " h B0 / 2 #

para 1H em magnetos normais (2,35 a 18,36 T) as frequncias

esto entre 100 e 800 MHz. Para 13C cerca de 1/4...
!-rays

10-10

X-rays UV VIS

10-8

IR

-wave

10-6 10-4
10-2
wavelenght (cm)

radio

100

102

NMR

Energia e frequncia
Constantes giromagnticas de alguns ncleos:

'0 = " B0 / 2 #

NMR

Istopo

" rad.s-1T-1

Freq a 11.74 T

1H

267,552*106

500,00

2H

41,066*106

76,753

13C

67,283*106

125,725

14N

19,338*106

36,132

17O

-36,281*106

67,782

10B

28,747*106

53,718

11B

85,847*106

160,420

19F

251,815*106

470,470

31P

108,394*106

202,606

23N

70,808*106

132,259

27Al

69,763*106

130,285

" razo magnetogrica

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Precesso
Rotaes, hertz e radianos...
A velocidade de precesso ou frequncia de Larmor
define-se como:

(0= 2 # '0

(0 = " B0 (radianos)

Associado a todos os ncleos (magnticos ou no) existe um

momento angular L
podemos imaginar os ncleos como pequenos
pies magnticos a rodar sobre si prprios

L!

NMR

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Precesso
Num campo magntico pode considerar-se que existem
duas foras a actuar sobre o ncleo. Uma que tenta
alinh-lo com B0 e outra que tenta manter o momento
angular.
!o

B0
L!

(0= 2 # '0

NMR

(0 = " B0 (radianos)

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Precesso
Os spins no se alinham com B0, independentemente da
sua orientao inicial

B0

Os spins precessam em torno de B0, no ngulo em que se

encontram quando colocados em B0.
Existem vrios campos magnticos a actuar sobre os spins. Um deles B0
que constante no tempo e responsvel pela precesso frequncia (0. Os
outros so flutuantes, devido anisotropia molecular e ao ambiente.

NMR

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Precesso
Os campos magnticos flutuantes criam as condies para que os spins
experimentem todas as orientaes possveis em relao a B0 num
determinado perodo de tempo.

B0

Orientaes a favor de B 0 , possuem uma energia

magntica menor e so favorecidas. Ao fim de um certo
tempo (relaxao longitudinal) desenvolve-se uma
magnetizao resultante (M0) na direco de B0.
NMR

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Magnetizao de equilbrio
Qual a origem da magnetizao de equilbrio ?
Se se decompuserem todos os vectores em z e <xy>
z

=
Mo

x
y

B0

A magnetizao resultante est alinhada com B0

NMR

A closer look at the interactions between magnetic

moment m and external magnetic field B0 :

NMR

B Volkman - MCW

Bulk magnetization
!

The macroscopic magnetization, Mo, is directly proportional to the

population difference (N - N), in which contributions from different s
precessing about B0 have been averaged:

!
!
!
!
!y
!
!

Mo

x
y

Bo

Bo

We can decompose each little in a z contribution and an

<xy> plane contribution. The components in the <xy> plane
are randomly distributed and cancel out. For the ones in z,
we get a net magnetization proportional to N - N.

!
!

There is an important difference between a and Mo. While

the former is quantized and can be only in one of two states
( or ), the latter tells us on the whole spin population. It
has a continuous number of states.
NMR

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Magnetizao/Excitao
Para produzir um sinal em RMN necessrio perturbar as
populaes
O sistema tem que absorver energia. A fonte de energia uma radiao
electromagntica oscilante, gerada por uma corrente alterna.
z

B1 = C * cos (!ot)

Mo
B1
y

i
bobine transmissora (y)

NMR

B0

E Cabrito - FCTUNL

Magnetizao/Excitao
z

B1 = C * cos (!ot)

Mo
B1
y

B0

i
bobine transmissora (y)

+!o

-!o

Uma variao linear em y uma combinao linear de dois campos

circulares em contra-rotao
NMR

B Volkman - MCW

Generating an NMR signal

!
When the frequency of an applied alternating current (B1 field) is o, we achieve
a resonant condition. The alternating magnetic field and Mo interact, there is a
torque generated on Mo, and the system absorbs energy :

!
!
!
!
!
!
!
!
!

Mo
B1
y

z
x

B1 off

!
!

(or off-resonance)

x
y

Since the system absorbed energy, the equilibrium of the

system was altered. We modified the populations of the N&

and N energy levels.

Again, keep in mind that individual spins flipped up or down

(a single quanta), but Mo can have a continuous variation.
NMR

Mxy

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Referenciais
Referencial do laboratrio e referencial rotatrio (rotating
frame)
O referencial anterior complicado de analizar, todo o sistema roda a
uma velocidade (0
A soluo adoptar um sistema de coordenadas que se move
velocidade (0. como se removessemos o efeito de B0
z

B0

Mxy

Mxy

!o

referencial do laboratrio

referencial rotatrio

Neste sistema de coordenadas Mxy no se move para fora da condio

de ressonncia (( de B1 exactamente igual frequncia do ncleo (0
NMR

Return of Mo to equilibrium (and detection)

!

B Volkman - MCW

In the absence of the external B1, Mxy will try to go back to Mo

(equilibrium) by restoring the same N / N distributiuon (relaxation)

Mxy returns to the z axis while precessing on the <xy> plane

!
!
!
!
!
!
!
!

Mxy

Mo

equilibrium...

The oscillation of Mxy generates a fluctuating magnetic field

which can be used to generate a current in a coil:
z

x
y

Receiver coil (x)

NMR

Mxy

record timedomain signal (FID)

FT

NMR signal

Free induction decay - FID

Que sinal podemos detectar na bobine de deteco depois
de colocar a magnetizao no plano <xy> ?
A amostra ir regressar ao equilbrio (z) precessando. No referencial
rotatrio a frequncia desta precesso ( - (0.
Mxy

!"#$%#&'($)*)+'#

!"#$%#&'($%)%*'#

"%)#

(#$"

"#

("

$%&#
$%'#

!#'"

$%(#
$%)#

!#&"

$#
$#

!#%"

$%*#

"#

"%*#

)#

)%*#

+#

+%*#

(#

(%*#

!$%)#
!$%(#

!#$"

!$%'#
!$%&#

!"
!"

!#)"

("

(#)"

$"

$#)"

*"

*#)"

%"

%#)"

!"#

tempo

tempo

( - (0 = 0

( - (0 > 0

Como a relaxao de M0 no plano <xy> exponencial a bobine

receptora detecta um sinal co-sinusoidal em decaimento.
NMR

Free induction decay - FID

Numa amostra real existem centenas de sistemas de spin
com frequncias diferentes de B 1 (frequncia de
referncia)

Como utilizmos um pulso de radiofrequncia que excitou todas as

frequncias, na bobine receptora detectamos um sinal que uma
combinao de todas essas frequncias - interferograma - Free
induction decay - FID
NMR

Free induction decay - FID

A transformada de Fourier do Free induction decay - FID permite obter
o espectro de RMN
s(t) = 1/2 #

! S(() e
--

i(t dt

S(() =

! s(t) e
--

-i(t dt

FT

FID - gerado por um pulso de

radiofrequncia que excitou todas
as frequncias. O sinal detectado
um interferograma, uma
combinao de todas essas
frequncias

Espectro - obtido a partir do FID

por uma operaao matemtica, a
transformada de Fourier

NMR

A Fourier Transform is used to

deconvolute the time domain signal

NMR spectrum

Frequency or Energy
NMR

E Cabrito - FCTUNL

Relaxao longitudinal
A recuperao da magnetizao ao longo do eixo z
chamada de relaxao longitudinal (ou spin-latice) e
corresponde ao restabelecimento das populaes de
equilbrio
z

y x

y x

T1 a constante temporal (de primeira ordem, 1/T1 ser a constante de

velocidade) para o processo de relaxao longitudinal (Mz = M0(1 - e-t/T1).
T1 no corresponde ao tempo que demora a recuperar a magnetizao.
NMR

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Relaxao transversal
A relaxao transversal (ou spin-spin) corresponde
perda da coerncia de fase no plano <xy> e
consequentemente da componente de magnetizao nesse
plano, Mxy.

tempo

T2 a constante temporal para o processo de relaxao transversal.

T2 est relacionada com a largura de linha detectada em RMN aps FT.
NMR

E Cabrito - FCTUNL

Mecanismos de relaxao
Relaxao longitudinal ou T1
Funciona para as componentes de magnetizao alinhadas com o eixo z
(Mz):
- perda de energia para o sistema sob a forma de calor
- acoplamento dipolar com outros spins, interaco com partculas
paramagnticas, etc...

Relaxao transversal ou T2
Actua nas componentes de magnetizao alinhadas no plano <xy>
(Mxy):
- as interaces spin-spin (J) retiram fase a Mxy
- imperfeies na homogeneidade do campo magntico (fanning out)
- como T1 tambm responsvel pela perda de Mxy, T2 nunca pode ser
superior a T1

NMR

E Cabrito - FCTUNL

Relaxao transversal
T2 longo

(#$"

("

!#'"

!#&"

!#%"

!#$"

!"

!#$" !#%" !#&" !#'"

("

(#$" (#%" (#&" (#'"

$"

$#$" $#%" $#&" $#'"

)"

)#$" )#%" )#&" )#'"

%"

!"

!#$" !#%" !#&" !#'"

("

(#$" (#%" (#&" (#'"

$"

$#$" $#%" $#&" $#'"

)"

)#$" )#%" )#&" )#'"

%"

!"
!"

!#$"

!#%"

!#&"

!#'"

("

relaxao lenta

(#$"

x
y

T2 curto

(#$"

("

!#'"

!#&"

!#%"

!#$"
!"

!#$" !#%" !#&" !#'"

("

(#$" (#%" (#&" (#'"

$"

$#$" $#%" $#&" $#'"

)"

)#$" )#%" )#&" )#'"

%"

!"

!#$" !#%" !#&" !#'"

("

(#$" (#%" (#&" (#'"

$"

$#$" $#%" $#&" $#'"

)"

)#$" )#%" )#&" )#'"

%"

!"
!"

!#$"

!#%"

!#&"

!#'"

("

tempo

NMR

(#$"

relaxao rpida

"#1/2 =

$T2*

Effect of molecular size

Rotates fast

Rotates more
slowly

10s-100s Hz !

a fewHz

NMR

We have a size limit in liquid NMR!

E Cabrito - FCTUNL

Desvio qumico
Se a cada tipo de ncleo corresponde uma frequncia (
qual a utilidade da RMN ?
nuvem
electrnica
ncleo

Bloc

Bef = B0 - Bloc

B0

campo magntico local

Campo magntico aplicado

NMR

E Cabrito - FCTUNL

Desvio qumico
Se a cada tipo de ncleo corresponde uma frequncia (
qual a utilidade da RMN ?

Bef = B0 - Bloc

nuvem
electrnica

ncleo

Bloc

Bef = B0 (1 - +)

campo magntico
local
Campo magntico
aplicado

B0

+ a blindagem magntica
afectado pela vizinhana do ncleo
(tipo de tomo, grupo funcional, etc)

NMR

B Volkman - MCW

Chemical shifts
!

If each type of nucleus has its characteristic o at a

certain magnetic field, why is NMR useful?

Depending on the chemical environment we have

variations on the magnetic field that the nuclei feels,
even for the same type of nuclei. It affects the local
magnetic field.

Beff = Bo - Bloc --- Beff = Bo( 1 - )

is the magnetic shielding of the nucleus. Factors

that affect it include neighboring atoms, aromatic
groups, etc., etc. The polarization of the bonds to the
observed nuclei are also important.

HO-CH2-CH3

As a crude example, ethanol looks like this:

high
field

low
field
o
NMR

Desvio qumico
Escalas e referncias diferentes para ncleos diferentes
1H

lcoois, protes
$ a cetonas

aromticos
e amidas

cidos e
aldedos

olefinas

alifticos

CH3

ppm
15

10

aromticos
alcenos conjugados

13C
C=O cetonas

0
TMS

H 3C

CH3, CH2, CH
alifticos

olefinas

150
C=O cidos
steres

100

80

50

CH3

CH3

ppm
210

Si

Tetrametilsilano - TMS

0
TMS

carbonos adjacentes a
lcoois e cetonas

NMR

B Volkman - MCW

The NMR scale (, ppm)

!

We can use the frequency scale as it is. The problem is that

since Bloc is a lot smaller than Bo, the range is very small
(hundreds of Hz) and the absolute value is very big (MHz).

We use a relative scale, and refer all signals in the spectrum

to the signal of a particular compound.

!
!
!
!

- ref

ppm (parts per million)

ref

The good thing is that since it is a relative scale, the in a

100 MHz magnet (2.35 T) is the same as that obtained for
the same sample in a 600 MHz magnet (14.1 T)

2,2-Dimethyl-2-silapentane- 5-sulfonate (DSS) is used as the 0

ppm chemical shift reference for 13C and 1H. Liquid NH3 is the
15N standard.
Lehninger, Nelson & Cox, 3rd ed.
NMR

NMR

B Volkman - MCW

1H

NMR of Proteins

150 residue protein has

~1000 1H signals
Different frequencies for
different methyls - due to
effects of 2 and 3
structure

methyl

Incomplete dispersion of
frequencies
Solution: separate
resonances in a second
frequency dimension amide
but how?

NMR

aliphatic

aromatic

Lehninger, Nelson & Cox, 3rd ed.

B Volkman - MCW

1H

NMR of Proteins

to obtain a structure we
need to identify every
peak in the spectrum
identify means relate the
frequency (ppm) to an
atom in the protein
once we identify the
frequency (ppm) of all
the atoms in our protein
we can determine a
structure

methyl

aliphatic

aromatic

amide

Lehninger, Nelson & Cox, 3rd ed.

NMR

How Can NMR Determine 3D

Structures?

NOE

Nuclear Overhauser effect (NOE) arises between

two 1H which are close in space (< 5), without
regard for covalent structure!
Proteins have many pairs of
nuclei in close
proximity which should give rise to NOE
correlations!

B Volkman - MCW

1 = 1x10-10 m

1H

Many of these 1H pairs will be from residues

which are far apart in the amino acid sequence
and contain tertiary information!

Hundreds of NOE restraints will severely restrict

the number of conformations which are
consistent with all the data!

Distance geometry and molecular dynamics

programs efficiently calculate structures from
NOE restraint data

NMR

1D NMR spectrum of a 10 kDa protein

Covalent environment dictates approximate range of chemical shifts

(HN, CH, CH3 etc.)
Non-covalent environment dictates where peak falls within that range
(type of adjacent AA, -helix, -sheet).
NMR

Amino acids

NMR

Amino acids

Covalent environment dictates approximate range of chemical shifts

(HN, CH, CH3 etc.)
Non-covalent environment dictates where peak falls within that range
(type of adjacent AA, -helix, -sheet).
NMR

Sample preparation
How do we prepare the sample ?
1. Dissolve in an adequate solvent (has to be soluble obviously!)

2. The sample has to be stable for 3-4 days minimum

3. Have to use a deuterated solvent (ex. 90%H2O+10%D2O)

to LOCK the spectrometer to correct for main field
fluctuations
Using 90% H2O we have to supress the
very strong water signal (110 M) to see the
protein signals (1 x 10-3 M)

0.5 ml of solution
Concentration ca. 1mM
5 mm

NMR

Sample preparation - the deuterium lock

Magnets used in high resolution NMR are not perfect and are prone to
drift for a variety of reasons.!

To compensate for this drift and to hold the magnetic field as stable as
possible the field-frequency lock was developed!

The lock unit is for all intents and purposes a self contained mini-NMR
which measures most often the resonance position of deuterium.!

As the field drifts the deuterium signal drifts and the lock follows this. The
drift in proton (or any other nucleus) signal can subsequently be adjusted!

Deuterated solvents are used which have the same properties as "light"
solvents and have substantially reduced proton signal (some solvents are
> 99.99% deuterated)!

Use at least 5% D2O

NMR

W Westler - UMadison

Sample preparation

(to reduce aggregation)

(to control pH)

contain unpaired electrons with very large magnetic moment - produces fast relaxation in nearby protons
NMR

W Westler - UMadison

Sample preparation

NMR

Sample amount

For 400l of a 1mM solution

NMR

NH resonances and chemical exchange

Lone pair of electrons available on N

Fast H2O NH exchange = no peak for NH2
Lone pair of electrons involved in peptide bond on peptide N

Slower H2O NH exchange = only loss of peak intensity in 10% D2O

NMR

NH resonances and chemical exchange

In 100% D2O, all NHs not invloved in stable H-bonds will eventually
exchange to ND - this is a way to detect H-bonding in proteins
NMR

NH resonances and chemical exchange

In 100% D2O:

!
!

Amino acids and small peptides - all NHs exchange to NDs.

Peptides and proteins - 1 backbone NH exchanges fast, the

rest eventually exchange.

!
!

In 9/1 H2O/D2O:

Proteins all backbone NHs except 1 seen

Amino acids and peptides (low pH) 1 NH can appear

NMR

pH and chemical exchange

Slow H2O NH exchange = no loss of peak intensity

Fast H2O NH
exchange = loss of
peak intensity

NMR

NMR spectrum of a 10 kDa protein

Covalent environment dictates approximate range of chemical shifts

Non-covalent environment dictates where peak falls within that range.

Note severe peak (resonance) overlap

NMR

NMR experiments - pulse sequences

Place sample in an external magnetic field (Bo)

Apply one or more radiofrequency pulses (B1) separated by carefully selected

delays. The sequence is repeated to increase SN and to remove artifacts via phase
cycling

Measure the frequency and intensity of the precessing signal, as well as other
parameters.

!
There are many different kinds of NMR experiments (pulse sequences)
!

Different pulse sequences are designed to measure different parameters

NMR

Information from NMR experiments

NMR Measurables

Information Obtained

precessional frequency
scale: chemical shift
units: ppm or Hz

local atomic environment

covalent
non-covalent

spin-spin coupling (J)

units: Hz

through-bond connections
between nuclei ( 3 bonds)
torsion angles: ,

nuclear Overhauser enhancement

(NOE)

through-space connections
between

spin relaxation rates

molecular motions, flexibility,

conformational exchange

residual dipolar couplings

relative orientation of
subunits, domains

peak intensities

number of contributing nuclei

NMR

B Volkman - MCW

Two basic types of internuclear correlations

Scalar ( J ) coupling: arises

between nuclear spins separated by
a small number of covalent bonds :
COSY, TOCSY, HMQC, HSQC

NOE
H 5 H

H
H

NMR

Nuclear Overhauser effect (NOE)

arises from dipolar interactions
between two 1H which are close in
space (<5), without regard for
covalent structure: NOESY

J
C

B Volkman - MCW

Scalar (J) Couplings

!

The energy levels of a nucleus will be affected by the spin state of nuclei nearby.
The two nuclei that show this are said to be coupled to each other. This manifests
in particular in cases were we have through bond connectivity:

!
!
!
!
!

1
13

H
C

three-bond

one-bond

Each spin now has two energy sub-levels depending on the state of the spin it is
coupled to:

!
!
!

!
!
!
!

J (Hz)

The magnitude of the separation is called the coupling constant (J) and has
units of Hz.
Coupling patterns are crucial to identify spin systems in a
molecule and to the determination of its chemical structure.
NMR

Constantes de acoplamento
Sistema de 1 ordem - &, >> J
O
CH3-C-O-CH2-CH3
# 4,5 ppm

# 1,5 ppm

cada 1H no grupo CH2 v quatro

estados possveis do CH3

cada 1H no grupo CH3 v trs

estados possveis do CH2
J # 7 Hz

%%%

%%

%%$ %$% $%%

$$% $%$ $$%

%$

$%
$$

$$$

CH2

J # 7 Hz

# 4,5 ppm

# 1,5 ppm

1:3:3:1

1:2:1

CH3

But, normally we do not see fine structure in our NMR spectra of proteins due to
their size (broad lines)
NMR

H3-C-O-CH2-CH3
# 4,5 ppm

# 1,5 ppm

J # 7 Hz

upo CH2 v quatro

cada 1H no grupo CH3 v trs
veis do CH3amino acid/small peptide
estados possveis do CH
protein
2
J # 7 Hz

%%

$%%
$$%

%$

$%
$$

# 4,5 ppm

# 1,5 ppm

1:3:3:1

1:2:1

# 4,5 ppm

CH3

# 1,5 ppm

But, normally we do not see fine structure in our NMR spectra of proteins due to
their size (broad lines)
NMR

B Volkman - MCW

Nuclear Overhauser Effect

Selectively saturate one 1H signal prior to normal 1D acquisition (~1-3 sec), allow
cross-relaxation to nearby spins - mediated by direct dipolar interactions
Difference spectrum reveals 1H within ~5
saturate
dec off
dec on

NOE

off on

Overlap makes selective saturation of only one resonance impossible => 1D

version normally of limited utility for proteins
Use in 2D or 3D mode to obtain structural information on proteins
NMR

1D Spectra for Amino acids

NMR

1D NMR

CH3

1D 1H spectrum of Alanine in 100% D2O

TSP

HOD

CH

NMR

1D NMR
1D

1H

spectrum of Alanine in 100% D2O

CH3 - 1:1

CH - 1:3:3:1

Area 1

Area 3

NMR

NMR spectrum of 100 amino acids

Covalent environment dictates approximate range of chemical shifts

Non-covalent environment dictates where peak falls within that range.

Note severe peak (resonance) overlap

NMR

How do we avoid overlap ?

1D

Fourier
Transform

2D NOESY 2D Fourier
Transform

t1

NMR

2D NMR
- Ideia proposta por J. Jeener em 1971 (Ampere International Summer
School, Yugoslavia)!
- Os grupos de Richard R. Ernst e Ray Freeman realizaram as
primeiras experincias (J.Chem.Phys. 1975)
Mtodos baseados no acoplamento de dipolos nucleares: interaces podem ser
escalares (atravs das ligaes) ou dipolares (atravs do espao).!
transferncia de magnetizao por permuta qumica

- HOMONUCLEARES 1H-1H: COSY, TOCSY e NOESY (EXSY)

- HETERONUCLEARES 1H-15N/13C: HSQC / HMQC
The Nobel Prize in Chemistry 1991
"for his contributions to the development of the methodology of high resolution nuclear
magnetic resonance (NMR) spectroscopy" Richard R. Ernst Switzerland
NMR

2D NMR - experiment
1D Delay then pulse then acquire
Preparation

2D Delay then pulse then incremented

Detection
- Delay during which the signal is
acquired (t2)

delay then mixing then acquire

Preparation

Evolution

t1
delay,pulse
Preparation
- Waiting time for system to
return to equilibrium
- Followed by one or more
pulses
Evolution

Detection

Mixing

Detection

delay,pulse

t2

Mixing
- Combination of delays and/or pulses
- magnetization transfer

2D = all correlations

- Evolution spin systems are not in

equilibrium
- Varies expt to expt.

can be seen in one

expt.

NMR

2D NMR - acquisition and processing

z

t1

x
90x

90x

Modulao da amplitude do
sinal variando t1
FT(t2)

FT(t1)

NMR

2D NMR - acquisition and processing

NMR

2D NMR

A BONUSregions in 2D
spectra provide protein
fingerprints

If 2D cross peaks overlap

go to 3D or 4D

NMR

Homonuclear 2D experiments

t1

spinlock

t1

tmix

COSY

TOCSY

J,J

NOESY

espacial

- Do origem a espectros caracterizados por uma diagonal (com simetria relativamente

diagonal)
- O tempo de mistura desenhado para a seleco de cada tipo de interaco entre
ncleos: transferncia de magnetizao baseada em acoplamento escalar ou interaco
dipolar
NMR

COSY - correlation spectroscopy

Ncleos A e X (JAX)
d1

t1

t1

t2

Double Quantum Filtered COSY

Perda de sensibilidade compensada
com a clarificao da regio diagonal

F1

Utilizado quando queremos supresso

de gua.

F2

Permite obter informao

acerca das constantes de
acoplamento
NMR

COSY spectrum
regies bem definidas
aumento resoluo relativamente a 1D

i-1

i+1

NMR

COSY correlations
Correlaes 3J - 3 ligaes qumicas s...

HS
CH2

O
C
H

N
H

HN
10

ppm

Cistena (C)

CH3

H 3C

NMR

CH

ppm

O
C
HN
Valina (V)

10
10

H H
ppm
ppm

H
00

COSY spectrum
Para identificar os desvio quimicos
todos de um amino acido temos que
andar em zonas com sobreposio
Amino acid 1
Proton

ppm

NH

9.25

3.36

1.57

0.58

i-1

i+1

NMR

TOCSY - total correlation spectroscopy

tmix
t1

spinlock

Pulso 180 refoca a evoluo dos desvios qumicos;

acoplamentos spin-spin mantm-se activos

B1

X
H
Os vectores esto locked
ao longo do eixo B1

CH2-CH-CH-O-CH-CH2

- A sequncia de mistura permite vrios saltos da magnetizao,

espalhando-a por um grupo de protes interligados por uma constante de
acoplamento J.
- transferncia de magnetizao baseada em acoplamento escalar
NMR

TOCSY correlations
HS
CH2

O
C

N
H

Cistena (C)

10

ppm
H

CH

ppm

C
HN

Valina (V)

10

NMR

2D NMR
2D TOCSY spectra of Alanine in 100% D2O

NMR

CH3

H 3C

HN

ppm

2D NMR
2D TOCSY spectra of Alanine in 100% D2O

NMR

2D NMR
2D TOCSY spectra of Alanine in 10% D2O

NMR

TOCSY patterns
Each type of amino acid has a
typical TOCSY pattern
Some amino acids have the
same pattern - SS of type AMX
(Y, D, F, H, N, and C)

NMR

TOCSY patterns
Each type of amino acid has a
typical TOCSY pattern
Some amino acids have the
same pattern - SS of type AMX
(Y, D, F, H, N, and C)

NMR

TOCSY patterns
9

NMR

TOCSY patterns

9
8
7

NMR

H
H
H
H

HN

TOCSY, COSY comparison

NOESY
NMR

TOCSY

COSY

NOESY

t1

tmix

Transferncia de magnetizao
atravs de acoplamento dipolar
-informao 3D

Informao acerca de ncleos d < 5

-tmix
N64
G125
H2C

COO-

H
H

NH2
H
H3C
N

NH3+

A25
G10

ANOE 1/rij6

NMR

NOESY, TOCSY, COSY comparison

NOESY
NMR

TOCSY

COSY

NMR resonance assignment strategies

!
Atribuio especfica das ressonncias; correlao entre os picos
do espectro de RMN e todos os 1Hs da protena

! Estratgias para a atribuio

! das ressonncias
!

NH S35

HA Q135

Definio das restries conformacionais; distncias entre protes

e ngulos de torso. Determinao de elementos da estrutura
secundria

!
Clculo da estrutura terceria

NMR

NMR resonance assignment strategies

!
Stage I:
Establish sequence-specific resonance assignments
which correlate NMR peaks with known 1 sequence
!
!
!
The sequence must be known prior to
establishing the assignments.
!
We are NOT using NMR to sequence the protein!!
!
!

NMR

NMR resonance assignment strategies

Strategy 1
Sample: no isotopic enrichment
NMR: 2D 1H-1H TOCSY and NOESY
8-10 kDa limit

!
!

Strategy 2
Sample: uniformly [99%,15N]-enriched protein
NMR: 3D 15N-resolved TOCSY and NOESY
12-14 kDa limit
Stragegy 3

Sample: uniformly [99%,13C/15N]-enriched protein

NMR: 3D triple-resonance experiments
20-25 kDa limit
Stategy 4

Sample: uniformly [90% 2H, 99% 13C/15N]-enriched protein

NMR: 3D/4D quadruple-resonance experiments
50-100 kDa limit
NMR

Assignment strategy 1
Protenas < 15kDa
- Sem marcao isotpica: at 10kDa

!
1- Identificao das ressonncias de cada amino cido (sistema de spin)

!
!

2- Atribuio sequencial e especfica

!
T! - L - G - S
!

R-G-S

1 2 3 4 5 6 7 !
R-G-S-T-L-G-S

Utilizao dos espectros TOCSY (COSY) e NOESY

NMR

Assignment strategy 1

i-1

i+1

2D TOCSY: intra-residue correlations between protons

2D NOESY: inter-residue correlations between protons
NMR

Assignment strategy 1

i-1

i+1

2D TOCSY: intra-residue correlations between protons

2D NOESY: inter-residue correlations between protons
NMR

Sequential assignment

NMR

Chemical shifts
Approximate Chemical Shift
Values for 1Hs in Different
Amino Acid Types
ballpark starting values:
NH: 6-10 ppm
H: 4-6 ppm
aromatic H: 6-8 ppm
CH2: 1-4 ppm
CH3: <1 ppm

NMR

Chemical shifts - BMRB database

chemical shifts

NMR

Chemical shifts - BMRB database

NMR

chemical shifts

Chemical shifts - BMRB database

NMR

Chemical shifts - BMRB

NMR

standard compounds

Chemical shifts - BMRB

standard compounds

NMR

Chemical shifts - BMRB

NMR

standard compounds

Chemical shifts - BMRB

alanine spectrum

NMR

Chemical shifts - BMRB

NMR

update display

Chemical shifts - BMRB

1D 1H spectrum

NMR

TOCSY patterns

Each type of amino acid has a

typical TOCSY pattern
Some amino acids have the
same pattern - SS of type AMX
(Y, D, F, H, N, and C)

NMR

TOCSY strip
HN:
8.1 ppm

These NMR peaks

are in the same
residue, and the
residue type is Val
Typical peak
pattern for Val in a
TOCSY spectrum

H:
H,:
1.9 ppm 0.9 ppm

H:
5.1 ppm

Conclusion we have a valine that has 1H chemical shifts of 8.1(HN),

5.1(H), 1.9(H) and 0.9(H) ppm - BUT which valine is it in the
sequence ??
NMR

Sequential assignment

CH

R1

i+1

CH

R2

Correlaes dentro do mesmo resduo aa

NMR

TOCSY
- definem-se as correlaes entre todos
os protes de um amino cido
- identificam-se os resduos por padres
caractersticos

AMX

Sequential assignment

2D TOCSY:
correlaes entre protes intra-resduo
2D NOESY:
correlaes entre protes inter-resduo
1

E-L-A-T-L-G-S
A - B - C!

!
!

Q - L - A!
E - L - A!
M-L-A

A = E!
B = L!
C=A

NMR

Sequential assignment

NMR

NOESY!
- atribuio sequencial dos aas: correlaes
entre aas consecutivos (e dentro do mesmo aa)!
- atribuio especfica ( necessrio conhecer a
sequncia primria)

Strategy 1 for establishing assignments

TOCSY+NOESY tells us that we have a V before an S - if that
combination appears only once in our 1o sequence we have a
sequence specific assignment - if it appears more than once we have
to go the next aa - if its a T then we know the assignment is T19-V20S21 - if its a H then we have H29-V30-S29 etc..

1 sequence

The combination of the NMR results with the known

primary sequence gives the resonance assignments.
NMR

Strategy 1 for establishing assignments

2D TOCSY

2D NOESY

through-bond
connections ( 3 bonds)

through-space
connections ( 5)

intra-residue
connections

inter-residue
connections

defines residue type

defines residue neighbors

Clusters of NMR peaks consistent with

peptide segments
+
known 1 sequence

!
Sequence-specific resonance assignments
NMR

1H resonance assignment table

the end result of Stage I (strategy 1)

NMR

Protein structure determination by NMR

!
Stage I:
Establish sequence-specific resonance assignments
!
YOU ARE HERE!
!
Stage II:
Define conformational restraints
(interproton distances, torsion angles)
Map 2 structure
!
Stage III:
Calculate and refine the 3 structure
NMR

Stage II - conformational restraints

NMR Measurable

!
1.
2.
!
!
!
3.

Information Obtained

NOE

!
1.

!
!
chemical shifts

!
2.

3J

!
3.

1H-1H

coupling
constants

interproton distances
(<5)
backbone dihedral
angles, secondary
structure
dihedral angles

NMR

Chemical shift index - CSI (H)

Diferenas dos desvios qumicos dos protes HA, relativamente aos valores random coil!

Valores > RC (cdigo= +1)

Folha beta!

Valores < RC (cdigo= -1)

Hlice alfa

NMR

13C

spectrum of a protein

NMR

Chemical shift index - CSI (C)

Wishart & Sykes (1994) J. Biomol. NMR 4, 171

Hlice

NMR

Folha

C, CO

+1

-1

-1

+1

-1

+1

Chemical shift index - CSI - Procedure H

Need sequential assignment of backbone Ha protons using standard 2-D or 3-D NMR techniques.!

(2) Using Table II carry out the following procedure for each residue in the protein:!
(a) If the Ha chemical shift is greater than the range given in Table II for that residue, mark a 1 beside it.!
(b) If the Ha chemical shift is less than the range given in Table II for that residue, mark a -1 beside it.!
(c) If the Ha chemical shift is within the given range in Table II for that residue, mark a 0 beside it.!

The above procedure defines the chemical shift index for each residue in the protein. Using these chemical
shift indices, we proceed to identify the secondary structures as follows:!

(3) Any dense grouping of four or more -1s not interrupted by a 1 is alpha-helix. Any dense grouping of
three or more 1s not interrupted by a -1 is a beta-strand. All other regions are designated as coil.!

(4) A minimum of three consecutive 1s is needed to define a beta-strand,and a minimum of four (not
necessarily consecutive) -1s is needed to define an alpha-helix. All remaining regions are defined as coil.!

(5) Termination points (at either end) of helices or P-strands can often be recognized by the first
appearance of chemical shift indices that are opposite in magnitude to those of the corresponding
secondary structure. In cases where this does not occur, the first appearance of two consecutive zerovalued chemical shift indices marks the termination point.

NMR

Chemical shift index - CSI - Procedure C

Need sequential assignment of backbone Ha protons using standard 2-D or 3-D NMR techniques.!

Using the 13C chemical-shift reference values in Table 2 carry out the following procedure for each residue in
the protein:!

(a) If the measured Ca chemical shift is greater than the range for that residue, mark a 1 beside it;!
(b) If the measured Ca chemical shift is less than the range for that residue, mark a -1 beside it;!
(c) If the measured Ca chemical shift is within the range for that residue, mark a 0 beside it.!

The above procedure defines the chemical-shift index for each residue in the protein. Using these chemicalshift indices for Ca and carbonyl carbons, one may identify the secondary structures as follows:!

(3) Any 'dense' grouping of four or more 1s not interrupted by a -1 is a helix. Any dense grouping of three or
more -1s not interrupted by a 1 is a b-strand. All other regions are designated as coil.!

(4) A minimum of three consecutive -1s is needed to define a b-strand, and a minimum of four 1s is needed to
define a helix. All remaining regions not identified as either helix or b-strand are defined as 'coil'.!

(5) Termination points (at either end) of helices or [3-strands can often be recognized by the first appearance of
chemical-shift indices that are opposite in magnitude to those of the corresponding secondary structure. In
cases where this does not occur, the first appearance of two consecutive zero-valued chemical-shift indices
marks the termination point.

NMR

Chemical shift ranges

1H

13C

AROM

NMR

J-couplings in proteins

1H-13C HSQC
13C

1H

NMR

1H-13C HSQC

NMR

Sequential and Medium-range NOEs identify -helices

Predictable NOE patterns (short interproton distances) correspond to regular
secondary structure elements!
Sequential, (i,i+3), (i,i+4) NOEs define
helices!
Long-range cross-strand NOEs define
sheets

NMR

2 structure from NOE patterns

- Podem ser identificados

atravs dos padres de NOE

NMR

2 structure from NOE patterns

NMR

2 structure from NOE patterns

i+3

i
i+4

NMR

Karplus relation - peptide torsion angles

Martin Karplus showed that J from
vicinal coupled 1H atoms depends
on the dihedral angle between the
protons. This relationship can be
approximated by the famous
Karplus equation:

J()

A, B, and C are empirically

derived parameters.

J couplings provide a semi-quantitative measure of molecular

conformation
NMR

Karplus relation - peptide torsion angles

NMR

NOE

3D structure from NOEs

H 5 H
Ca
Pro 21

N
Met 12

C
NMR

3D structure from NOEs

N64
G125
H2C

COO-

H
H

NH2
H
H3C
N

NH3+

G10

A25

We used the region in the red box to identify sequential correlations

NMR

Brian Volkman - MCW

3D structure from NOEs

ANOE = C. 1/(rij6)

N64
G125
H2C

COO-

H
H

NH2
H
H3C
N

NH3+

A25

G10

NH G10 CH3 A25 4.0

CH3 A25 CH2 N64 4.5
CH2 N64 HA G125 3.0
NH G10 HA G125 2.5

Now we use the intensity of each cross peak in the whole NOESY (2D or
3D) spectrum to identify every cross peak - List of distances between
protons in the protein.
NMR

NOESY
t1

tmix

ANOE = C. 1/(rij6)
Transferncia de magnetizao
atravs de acoplamento dipolar

Informao acerca de ncleos d < 5

N64
G125
H2C

COO-

H
H

NH2
H
H3C
N

NH3+

G10

NH G10 CH3 A25 4.0

A25

Strong
1.8 4.5
- 2.7
CH3NOE
A25 CH2 N64

Medium NOE
1.8 - 3.3
CH2 N64 HA G125 3.0
Weak NOE
1.8 - 5.0
NH G10 HA G125 2.5

NMR

Integrao de todos os picos: !

programa SPARKY (XEASY, CARA)

NOESY
Informao acerca de ncleos d < 5
46 LYS !
HN
HN
HN
HN
HN
HA
HA
HA
HA
HA
HB2
HB2
HB2
HB3
HB3
QG
47 SER !
HN
HN
48 GLU !
HN
HN
HN
HN
HN
HN
HA
HA
HA
HB2
49 PHE !
HN
HN
HN
NMR HA
HB3
QD

46 LYS HB2
46 LYS HB3
46 LYS QG
46 LYS QD
47 SER HN
46 LYS HB3
46 LYS QD
48 GLU HN
49 PHE HN
49 PHE QD
46 LYS QE
46 LYS QZ
47 SER HN
46 LYS QE
46 LYS QZ
46 LYS QZ

3.43
3.37
5.51
6.88
3.42
3.27
6.88
3.95
4.29
6.10
3.81
5.82
4.20
6.72
4.99
7.32

!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!

47 SER HB2
48 GLU HN

3.27
3.18

48 GLU HB2 3.11

48 GLU HB3 3.58
48 GLU HG2 4.48
48 GLU HG3 3.70
49 PHE HN
2.80
49 PHE QD
6.66
48 GLU HB3 2.96
48 GLU HG3 3.21
49 PHE HN
3.33
49 PHE QD
7.62

!
!
!
!
!
!
!
!
!
!

49 PHE HB2
49 PHE HB3
50 GLU HN
50 GLU HN
50 GLU HN
50 GLU HN

!
!
!
!
!
!

3.55
3.30
4.17
2.40
3.39
6.35

Protein structure determination by NMR

Stage I:
Establish sequence-specific resonance assignments
!

!
Stage II:
Define conformational restraints
(interproton distances, torsion angles)
Map 2 structure
!
YOU ARE HERE!
!
Stage III:
Calculate and refine the 3 structure
NMR

Stage III - calculate and refine the structure

Hybrid distance geometry/simulated annealing (DG/SA)
DG uses NOESY-derived interproton distances as input,
calculates 3D maps consistent with input distances;
XPLOR/CNS, DISGEO/DGII, TINKER

Simulated annealing from random molecular coordinates

Incorporates input restraints into a energy function,
Surveys the potential energy of system to find global minimum
Programs that implement SA: XPLOR/CNS, others

Torsion angle refinement methods

Finds backbone , angles most consistent with restraints
Programs that implement this approach: DIANA, DYANA, CYANA

!
All of these calculations are performed with a computer
using the experimental restraints from Stage II as input.
NMR

Sumilated annealing
Programas que utilizam diferentes mtodos para calcular uma
famlia de estruturas (confrmeros) : XPLOR, DYANA, CYANA
cadeia polipeptdica numa conformao ao acaso

Utilizao de mtodos de mecnica clssica de

minimizao da energia total do sistema e.g.
Simulated Annealing. Incorpora as restries
experimentais numa funo de energia

Energy

r R

Simulao do movimento dos tomos em condies de aquecimento.

A protena aquecida provocando movimentos moleculares.
Depois arrefecida lentamente, de maneira a minimizar a energia.
NMR

Structure determination - the problem

a mathematical calculation that converts a table of
distances into a map or structure
46 LYS !
HN
HN
HN
HN
HN
HA
HA
HA
HA
HA
HB2
HB2
HB2
HB3
HB3
QG
47 SER !
HN
HN
48 GLU !
HN
HN
HN
HN
HN
HN
HA
HA
HA
HB2
49 PHE !
HN
HN
HN
HA
HB3
QD
50 GLU !
HN
HN
HN
HN
HN
HA
HG2
HG3

46 LYS HB2
46 LYS HB3
46 LYS QG
46 LYS QD
47 SER HN
46 LYS HB3
46 LYS QD
48 GLU HN
49 PHE HN
49 PHE QD
46 LYS QE
46 LYS QZ
47 SER HN
46 LYS QE
46 LYS QZ
46 LYS QZ

3.43
3.37
5.51
6.88
3.42
3.27
6.88
3.95
4.29
6.10
3.81
5.82
4.20
6.72
4.99
7.32

47 SER HB2
48 GLU HN

3.27
3.18

48 GLU HB2 3.11

48 GLU HB3 3.58
48 GLU HG2 4.48
48 GLU HG3 3.70
49 PHE HN
2.80
49 PHE QD
6.66
48 GLU HB3 2.96
48 GLU HG3 3.21
49 PHE HN
3.33
49 PHE QD
7.62
49 PHE HB2
49 PHE HB3
50 GLU HN
50 GLU HN
50 GLU HN
50 GLU HN

3.55
3.30
4.17
2.40
3.39
6.35

!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!

+
!

!
!
!
!
!
!
!
!
!
!

MET 1 LYS LYS TYR VAL CYSS THR VAL CYSS GLY!
TYR GLU TYR ASP PRO ALA GLU GLY ASP PRO!
ASP ASN GLY VAL LYS PRO GLY THR SER PHE!
ASP ASP LEU PRO ALA ASP TRP VAL CYSS PRO!
VAL CYSS GLY ALA PRO LYS SER GLU PHE GLU!
ALA ALA

!
!
!
!
!
!

Output: map of US

Input: intercity distance table

50 GLU HB2 3.52
50 GLU HB3 4.07
50 GLU HG2 3.76
50 GLU HG3 3.98
51 ALA HN
4.48
51 ALA HN
2.74
51 ALA HN
4.20
51 ALA HN
3.30

!
!
!
!
!
!
!

For NMR data:

Input:
1H - 1H distance table

Output:
structure of molecule

NMR

Cyana structure calculation

Note: many
structures have
similar final target
function energies!

In NMR we always
have a family of final
structures
NMR

Brian Volkman - MCW

Cyana structure calculation

30 TAD structures !
w/disulfide bond only

Assign NOEs, generate !

distance constraints

Calculate 60
structures with !
TAD (DYANA)

30 TAD structures !
w/disulfide and !
NOE restraints

NMR

Distance restraints

experimental distance
constraint

NMR

Distance restraints
Restries de distncias
experimentais

Numero de restries:
definio da estrutura

A 322 NOEs
B 657

Concluso: quanto mais

restries experimentais
melhor!

C 747
D 809

NMR

Distance restraints
Para aumentar o n de restries
(NOEs) podemos:

!
1. Aumentar a concentrao da
amostra
2. Aumentar o n de scans na
experiencia NOESY
3. Fazer o espectro a um campo
magntico maior

!
Neste maneira vamos aumentar o S/N
do espectro e conseguimos ver mais
sinais NOESY

NMR

NMR family
Cadeia principal

Estrutura de RMN representada

por um grupo de estruturas
sobrepostas (10-30)
RMSD (root mean square deviation)
entre os vrios modelos serve para
determinar a convergncia no
clculo das estruturas (preciso)

R=

(1/N) (ri ri)2

i=1

Boas estruturas: RMSD(BB)< 1

Zonas indefinidas devidas a

- Falta de restries
ou
- Flexibilidade Molecular

Cadeias laterais

Determinao de parmetros de
relaxao (T1 ou T2), permite
detectar mobilidade de cada
resduo

NMR

Zonas indefinidas devidas a

NMR family

- Falta de restries
ou
- Flexibilidade Molecular
Determinao de parmetros de relaxao
(T1 ou T2), permite detectar mobilidade de
cada resduo

NMR

Structure visualisation
Ribbon

Sobreposio de 15 estruturas

Com todas
as ligaes

Sobreposio
indentifica zonas
pouco definidas

S cadeia
principal

NMR

Structure determination by NMR

1970

1980

RMN
transformada
de Fourier

1990

2000

Ressonncia
Tripla

NOESY,
TOCSY

RDCs

TROSY

RMN 2D
Atribuio
proteinas 1H/
estruturas 3D

Atribuio
de protenas
13
C/15N

Biologia Molecular
Protenas
recombinantes,
expresso e
marcao isotpica
Tamanho da
protena

NMR

60aa

160aa

260aa

Mtodos
de
aquisio
rpida
Deteco
13
C para
Bio-RMN

Structure determination by NMR

NMR

Structure determination by NMR

Backbone conformation from chemical

shifts (Chemical Shift Index- CSI): ,

!
Distance restraints from NOEs
!
Hydrogen bond restraints
!

Backbone and side chain dihedral angle

restraints from scalar couplings

Orientation restraints from residual

dipolar couplings

CYANA / XPLOR

NMR

Protein structure determination by NMR

Stage I:
Establish sequence-specific resonance assignments
!
!
Stage II:
Define conformational restraints
(interproton distances, torsion angles)
Map 2 structure
!
!
Stage III:
Calculate and refine the 3 structure
!
YOU ARE HERE!
NMR

Assessing the quality of NMR structures

Quality of experimental data!
- choice of assignment strategy; appropriate for Mwt?!
- assessment of ambiguity, methods to circumvent it!
- number of conformational restraints per residue!

Agreement of calculated structures with experimental data!

- distance and torsion angle violations: number, sizes!

Agreement within the NMR ensemble (precision)!

- root mean square deviations (RMSD)!

Agreement of structures with a priori knowledge of molecular!

- geometry (stereochemical quality)!
- covalent bond lengths, bond angles, planarity, etc.!

Agreement of structures with a priori knowledge of proteins!

- Ramachandran plot, homology!

Agreement of structures with other biochemical data!

- limited proteolysis, x-ray crystal structures
NMR

NMR v X-ray Crystallography

Advantages of NMR
!Can be performed in the solution-state
!Structures in solution may be more physiologically relevant
!Some proteins do not give diffraction-quality crystals!
!Provides dynamics and other information: internal mobility,
flexibility, order-disorder, hydrogen exchange rates, pKa values,
binding constants, conformational exchange rates

Disadvantages of NMR
!Molecular weight limitations: ~50 kDa for complete structure
determination ~100 kDa for local or partial analysis
!Stable-isotope enrichment usually required: need efficient bacterial
expression system
!Eukaryotic expression largely impractical, expensive
!Structure determination methods more time consuming, difficult

NMR

NMR versus X-ray

The pros of NMR techniques!
! !
! !
! !
! !
! !
! !
! !

Closer to biological conditions!

Can provide information on dynamics and identify individual side-chain motion!
Secondary structure can be derived from limited experimental data!
Free from artifacts resulting from crystallization!
Can be used to monitor conformational change on ligand binding!
Solution conditions can be explicitly chosen and readily changed, e.g. pH, temperature, etc.!
Useful for protein-folding studies.!

The cons of NMR techniques!

! !
! !
! !
! !
! !
! !
! !

Requires concentrated solution - therefore danger of aggregation!

Currently limited to determination of relatively small proteins!
Surface residues generally less well defined than in X-ray crystallography!
The distinction between flexibility and lack of data is not always easy!
Produces an ensemble of possible structures rather than one model!
Conformational variability can make interpretation difficult!
Complete structure determination required if homology is less than 60 percent sequence
identity

NMR versus X-ray

The pros of X-ray crystallography!
! !
! !
! !
! !

Well-established technique!
More mathematically direct image construction!
Raw-data processing highly automated!
Mutants,different ligands and homologous structures (as low as 25 percent sequence identity)
readily compared by difference Fourier techniques!
! ! Large molecules and assemblies can be determined, e.g. virus particles!
! ! Surface water molecules relatively well defined!
! ! Produces a single model that is easy to visualize and interpret!

The cons of X-ray crystallography!

! !
! !
! !
! !
! !
! !
! !
! !
! !

Protein has to form stable crystals that diffract well!

Need heavy-atom derivatives that form isomorphous crystals!
Crystal production can be difficult and time consuming, and often impossible!
Unnatural, non-physiological environment!
Difficulty in apportioning uncertainty between static and dynamic disorder!
Surface residues may be influenced by crystal packing!
May not wholly represent structure as it exists in solution!
Less useful for large flexible modular proteins!
Model represents a time-averaged structure where details of mobility are unresolved!