unmr

NMR

Brian J Goodfellow!Departamento de Quimica!Universidade de Aveiro!Aveiro 3810-193!Email: [email protected]

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Protein structure determination by NMR

NMR

NMR - Bibliography

NMR

- Teng, Q. “Structural Biology: Practical NMR applications” Springer Science, USA (2005)

- Evans, J.N.S. “Biomolecular NMR Spectroscopy”, Oxford University Press (1995) - Wüthrich, 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. “Ressonância Magnética Nuclear. Fundamentos e aplicações”, Fundação 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 - Bibliography

NMR

Nobel prize - Physics 1952� � Discovery of the NMR effect��

Felix Bloch!Stanford,USA

Edward Mills Purcell,!Harvard, USA

NMR - Nobel Prizes

NMR

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. Wüthrich - ETH

NMR - Nobel Prizes

NMR

Nobel prize - Medicine 2003�� NMR Imaging� ��

Lauterbur - USA Mansfield - UK

NMR - Nobel Prizes

NMR

Principais aplicações RMN!!Elucidação estrutural!! Produtos naturais! ! Química orgânica. Ferramenta analítica de eleição dos químicos de ! síntese!Estudo de processos dinâmicos!! Cinética de reacções! ! Estudo de equilíbrio (químico ou estrutural)! Estudos estruturais (tridimensionais)!! proteínas DNA/RNA. Complexos de proteínas com DNA/RNA! Drug design - structure activity relationship (SAR) por RMN!Medicina - Magnetic Resonance Imaging (MRI, fMRI)

NMR DQ tem maior numero de espctrómetros RMN no país - 5

probe

300MHz - service

400MHz - solid state

500MHz - metabolomics, LCNMR

500MHz - natural products (cryo probe) 700MHz - solids, proteins

NMR

O espectro de RMN

Espectro de protão do etanol

1H, ! desblindado blindado

campo baixo campo alto

-CH2- CH3

-OH + H2O

Desvio químico (!), constante de acoplamento (J), largura de linha e intensidade

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NMR

Fundamentos

SPIN

RMN - Detecta a absorção de radiofrequências (radiação electromagnética) por certos núcleos numa molécula

Para descrever o fenómeno na totalidade é necessária alguma (muita mesmo) mecânica quântica

Ao contrário da massa atómica e da carga o spin não tem equivalente macroscópico, simplesmente existe...

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NMR

Fundamentos Só os núcleos com número quântico de spin (I) ! 0 podem absorver/emitir radiação electromagnética

massa atómica e número atómico par, I = 0 (12C, 16O)

massa atómica par e número atómico impar, I = inteiro (14N, 2H, 10B)

massa atómica impar, I = meio-inteiro (1H, 13C, 15N, 31P)

Os estados de spin de um núcleo (m) estão quantizados:

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

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I = 0

I = nº inteiro

I = 1/2

Ver tabelas para tirar I

NMR

Fundamentos

é um vector que dá a direcção e a magnitude do “magneto nuclear”

estes núcleos só podem existir em dois estados de spin

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

Momento magnético nuclear (µ):

m = 1/2, - 1/2

µ = " I h / 2 #! " razão magnetogírica

h constante de Plank

µ

L!

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NMR

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!!!!!!!!• 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.

Bo

= γ h / 4π

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NMR

Energia e populações Quanto maior B0, maior a diferença de energia

E

"E = " h B0 / 2 #

B0

$

% %

$

B0 &E

%$

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NMR

Energia e populaçõesQuanto maior B0 maior a diferença de energia.

A razão das populações dos dois estados depende de "E e pode ser calculada através da distribuição de Boltzmman

N$ /N% = e"E/kT

A "E para 1H a 400 MHz (B0 = 9.4 T) é 3.8 " 10-5 kcal/mol

N$ /N% = 1.000064

num milhão de spins só há uma diferença de 64A RMN é uma técnica muito pouco sensível (pelo menos quando

comparada com IV ou UV)

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NMR

Energia e sensibilidade Núcleos com " elevado irão absorver/emitir mais energia e

como tal serão mais sensíveis. A sensibilidade é proporcional a N$ /N% e ao fluxo magnético

da bobine e ambos dependem de ".

No total a sensibilidade depende de "3

1H é # 64 vezes mais sensível que 13C só devido a "

" 13C = 6,728 rad / G

" 1H = 26,753 rad / G

se se considerar também a abundância natural do 13C (# 1%) verifica-se que 13C é 6400 vezes menos sensível que 1H

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NMR

Useful nuclei such as 15N, 13C are rare

Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance g/107 rad T-1s-1 MHz (2.3 T magnet)

1H 1/2 99.985 % 26.7519 100.000000 2H 1 0.015 4.1066 15.351 13C 1/2 1.108 6.7283 25.145 14N 1 99.63 1.9338 7.228 15N 1/2 0.37 -2.712 10.136783 17O 5/2 0.037 -3.6279 13.561 19F 1/2 100 25.181 94.094003 23Na 3/2 100 7.08013 26.466 31P 1/2 100 10.841 40.480737 113Cd 1/2 12.26 -5.9550 22.193173

NMR


Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance g/107 rad T-1s-1 MHz (2.3 T magnet)

1H 1/2 99.985 % 26.7519 100.000000 2H 1 0.015 4.1066 15.351 13C 1/2 1.108 6.7283 25.145 14N 1 99.63 1.9338 7.228 15N 1/2 0.37 -2.712 10.136783 17O 5/2 0.037 -3.6279 13.561 19F 1/2 100 25.181 94.094003 23Na 3/2 100 7.08013 26.466 31P 1/2 100 10.841 40.480737 113Cd 1/2 12.26 -5.9550 22.193173

NMR


Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance (Ɣ/107) rad T-1s-1 MHz (2.3 T magnet)

1H 1/2 99.985 % 26.7519 100.000000 2H 1 0.015 4.1066 15.351 13C 1/2 1.108 6.7283 25.145 14N 1 99.63 1.9338 7.228 15N 1/2 0.37 -2.712 10.136783 17O 5/2 0.037 -3.6279 13.561 19F 1/2 100 25.181 94.094003 23Na 3/2 100 7.08013 26.466 31P 1/2 100 10.841 40.480737 113Cd 1/2 12.26 -5.9550 22.193173

NMR

Energia e populações

E = - µ . B0

momento magnético µ alinhado com o campo magnético, B0

!

momento magnético µ alinhado contra o campo magnético, B0

!

A energia de um spin num campo magnético vai depender do campo magnético, B0, e do momento magnético µ!

B0 µ

B0 µ

"E = " h B0 / 2 #

E$ = - " h B0 / 4 #

E% = " h B0 / 4 #

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NMR

Energia e frequência A energia está relacionada com a frequência...

'0 = " B0 / 2 #

para 1H em magnetos normais (2,35 a 18,36 T) as frequências estão entre 100 e 800 MHz. Para 13C cerca de 1/4...

"E = h '0

"E = " h B0 / 2 #

!-rays X-rays UV VIS IR µ-wave radio

10-10 10-8 10-6 10-4 10-2 100 102

wavelenght (cm)

NMR

Energia e frequência Constantes giromagnéticas de alguns núcleos:

" razão magnetogírica

Isótopo " 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

'0 = " B0 / 2 #

NMR

PrecessãoRotações, hertz e radianos...

(0 = " B0 (radianos)

Associado a todos os núcleos (magnéticos ou não) existe um momento angular L

(0= 2 # '0

A velocidade de precessão ou frequência de Larmor define-se como:

µ

L!

podemos imaginar os núcleos como pequenos piões magnéticos a rodar sobre si próprios

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NMR

Precessão

(0 = " B0 (radianos)(0= 2 # '0

Num campo magnético pode considerar-se que existem duas forças a actuar sobre o núcleo. Uma que tenta alinhá-lo com B0 e outra que tenta manter o momento angular.

!o

µ

L!

B0

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NMR

PrecessãoOs spins não se alinham com B0, independentemente da sua orientação inicial

B0

Os spins precessam em torno de B0, no ângulo em que se encontram quando colocados em B0.

Existem vários campos magnéticos a actuar sobre os spins. Um deles é B0

que é constante no tempo e responsável pela precessão à frequência (0. Os outros são flutuantes, devido à anisotropia molecular e ao ambiente.

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NMR

Precessão

B0

Orientações a favor de B0, possuem uma energia magnética menor e são favorecidas. Ao fim de um certo tempo (relaxação longitudinal) desenvolve-se uma magnetização resultante (M0) na direcção de B0.

Os campos magnéticos flutuantes criam as condições para que os spins “experimentem” todas as orientações possíveis em relação a B0 num determinado período de tempo.

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NMR

Magnetização de equilíbrio

B0

Qual a origem da magnetização de equilíbrio ?

Se se decompuserem todos os vectores em z e <xy>

y

x

z =

z z

x

y

= “0”

A magnetização resultante está alinhada com B0

Mo

z

x

y

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NMR

A closer look at the interactions between magnetic moment m and external magnetic field B0 :

NMR

Bulk magnetization !• The macroscopic magnetization, Mo, is directly proportional to the population difference (Nα - Nβ), in which contributions from different µs precessing about B0 have been averaged: !!!!!!!•We can decompose each little µ 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 Nα - Nβ. !!

• There is an important difference between a µ and Mo. While the former is quantized and can be only in one of two states (α or β), the latter tells us on the whole spin population. It has a continuous number of states.

Mo

y

x

z

x

y

z

Bo Bo

B Volkman - MCW

NMR

Magnetização/Excitação

B0

Para produzir um sinal em RMN é necessário perturbar as populações

O sistema tem que absorver energia. A fonte de energia é uma radiação electromagnética oscilante, gerada por uma corrente alterna.

B1 = C * cos (!ot)

Mo

z

i

B1

y

bobine transmissora (y)

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NMR

Magnetização/Excitação

B0

Mo

z

i

B1

y

B1 = C * cos (!ot)

bobine transmissora (y)

+ =

+!o -!

o

x x x

y y y

Uma variação linear em y é uma combinação linear de dois campos circulares em contra-rotação

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NMR

Generating an NMR signal !• When the frequency of an applied alternating current (B1 field) is ωo, we achieve a resonant condition. The alternating magnetic field and Mo interact, there is a torque generated on Mo, and the system absorbs energy : !!!!!!!!!• Since the system absorbed energy, the equilibrium of the system was altered. We modified the populations of the Nα&

and Nβ energy levels. !• Again, keep in mind that individual spins flipped up or down (a single quanta), but Mo can have a continuous variation.

B1 off… !!

(or off-resonance)

Mo

z

xB1

z

x

Mxyy y

ωo

ωo

B Volkman - MCW

NMR

Referenciais

O referencial anterior é complicado de analizar, todo o sistema roda a uma velocidade (0

Referencial do laboratório e referencial rotatório (rotating frame)

A solução é adoptar um sistema de coordenadas que se move à velocidade (0. É como se removessemos o efeito de B0

B0

z

x

Mxy

z

x

Mxy

y !o

referencial do laboratório referencial rotatório

Neste sistema de coordenadas Mxy não se move para fora da condição de ressonância (( de B1 é exactamente igual à frequência do núcleo (0

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NMR

Return of Mo to equilibrium (and detection) !• In the absence of the external B1, Mxy will try to go back to Mo (equilibrium) by restoring the same Nα / Nβ distributiuon (relaxation) !

• Mxy returns to the z axis while precessing on the <xy> plane !!!!!!!!The oscillation of Mxy generates a fluctuating magnetic field which can be used to generate a current in a coil:

z

x

Mxyy

z

x

y

z

x

Mxy

Receiver coil (x)

y

Mo

⇒ NMR signal

equilibrium...

ωo

ωo

⇒ record time-domain signal (FID)

t

FTω

B Volkman - MCW

NMR

Free induction decay - FIDQue sinal podemos detectar na bobine de detecção depois de colocar a magnetização no plano <xy> ?

A amostra irá regressar ao equilíbrio (z) precessando. No referencial rotatório a frequência desta precessão é ( - (0.

Como a relaxação de M0 no plano <xy> é exponencial a bobine receptora detecta um sinal co-sinusoidal em decaimento.

tempo

!"

!#$"

!#%"

!#&"

!#'"

("

(#$"

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

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

( - (0 = 0

!"#

!$%&#

!$%'#

!$%(#

!$%)#

$#

$%)#

$%(#

$%'#

$%&#

"#

"%)#

$# $%*# "# "%*# )# )%*# +# +%*# (# (%*#

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

tempo

( - (0 > 0

NMR

Free induction decay - FIDNuma amostra real existem centenas de sistemas de spin com frequências diferentes de B1 (frequência de referência)

Como utilizámos um pulso de radiofrequência que excitou todas as frequências, na bobine receptora detectamos um sinal que é uma combinação de todas essas frequências - interferograma - Free induction decay - FID

NMR

Free induction decay - FIDA transformada de Fourier do Free induction decay - FID permite obter o espectro de RMN

FID - gerado por um pulso de radiofrequência que excitou todas as frequências. O sinal detectado é um in ter ferograma, uma combinação de todas essas frequências

Espectro - obtido a partir do FID por uma operaçao matemática, a transformada de Fourier

s(t) = 1/2 # ! S(() ei(t dt

-

--

S(() = ! s(t) e-i(t dt

-

--

FT

NMR

A Fourier Transform is used to deconvolute the time domain signal

Frequency or Energy

NMR spectrum

NMR

Relaxação longitudinalA recuperação da magnetização ao longo do eixo z é chamada de relaxação longitudinal (ou spin-latice) e corresponde ao restabelecimento das populações de equilíbrio

x y

z

x y

z

x y

z

x y

z

T1 é a constante temporal (de primeira ordem, 1/T1 será a constante de velocidade) para o processo de relaxação longitudinal (Mz = M0(1 - e-t/T1). T1 não corresponde ao tempo que demora a recuperar a magnetização.

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NMR

Relaxação transversalA relaxação transversal (ou spin-spin) corresponde à perda da coerênc ia de fase no p lano <xy> e consequentemente da componente de magnetização nesse plano, Mxy.

T2 é a constante temporal para o processo de relaxação transversal. T2 está relacionada com a largura de linha detectada em RMN após FT.

x

y

x

y

tempo

x

y

x

y

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NMR

Mecanismos de relaxaçãoRelaxação longitudinal ou T1

Funciona para as componentes de magnetização alinhadas com o eixo z (Mz):- perda de energia para o sistema sob a forma de calor- acoplamento dipolar com outros spins, interacção com partículas paramagnéticas, etc...

Relaxação transversal ou T2

Actua nas componentes de magnetização alinhadas no plano <xy> (Mxy):- as interacções spin-spin (J) retiram fase a Mxy

- imperfeições na homogeneidade do campo magnético (fanning out)- como T1 também é responsável pela perda de Mxy, T2 nunca pode ser superior a T1

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NMR

Relaxação transversal

x

y

x

y

tempo

x

y!"

!#$"

!#%"

!#&"

!#'"

("

(#$"

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

!"

!#$"

!#%"

!#&"

!#'"

("

(#$"

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

T2 curto

relaxação rápida

!"

!#$"

!#%"

!#&"

!#'"

("

(#$"

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

!"

!#$"

!#%"

!#&"

!#'"

("

(#$"

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

T2 longo

relaxação lenta"#1/2 =

1

$T2*

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NMR

a fewHz 10’s-100’s Hz !

Rotates fast Rotates more slowly

We have a size limit in liquid NMR!

Effect of molecular size

NMR

Desvio químicoSe a cada tipo de núcleo corresponde uma frequência (

qual a utilidade da RMN ?

Campo magnético aplicado

B0

núcleo

nuvem electrónica

campo magnético local

Bloc

Bef = B0 - Bloc

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NMR

Desvio químicoSe a cada tipo de núcleo corresponde uma frequência (

qual a utilidade da RMN ?

Bef = B0 - Bloc

Bef = B0 (1 - +)

+ é a blindagem magnéticaB0

Bloc

Campo magnético aplicado

núcleo

nuvem electrónica

campo magnético local

afectado pela vizinhança do núcleo (tipo de átomo, grupo funcional, etc)

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NMR

Chemical shifts !

• If each type of nucleus has its characteristic ωo 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 - σ ) !• σ 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. !• As a crude example, ethanol looks like this: HO-CH2-CH3

ωo

low field

high field

B Volkman - MCW

NMR

Desvio químicoEscalas e referências diferentes para núcleos diferentes

CH3

Si

CH3

CH3H3C

Tetrametilsilano - TMSppm

50 150 100 80 210 0

TMS

0

TMS

ppm

2 10 7 5 15

C=O cetonas

C=O ácidosésteres

aromáticosalcenos conjugados

olefinasCH3, CH2, CH

alifáticos

carbonos adjacentes a álcoois e cetonas

ácidos e aldeídos

aromáticos e amidas

olefinas

álcoois, protões $ a cetonas

alifáticos

1H

13C

NMR

Lehninger, Nelson & Cox, 3rd ed.

The NMR scale (δ, 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. !!!!• The good thing is that since it is a relative scale, the δ 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.

ω - ωref δ = ppm (parts per million) ωref

B Volkman - MCW

NMR

NMR

1H NMR of Proteins

amidearomatic

methyl


aliphatic

● 150 residue protein has ~1000 1H signals

● Different frequencies for different methyls - due to effects of 2˚ and 3˚ structure

● Incomplete dispersion of frequencies

● Solution: separate resonances in a second frequency dimension - but how?

B Volkman - MCW

NMR

1H NMR of Proteins

amidearomatic

methyl


aliphatic

● 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

B Volkman - MCW

NMR

How Can NMR Determine 3D Structures?

• Nuclear Overhauser effect (NOE) arises between two 1H which are close in space (< 5Å), without regard for covalent structure!

• Proteins have many pairs of 1H nuclei in close proximity which should give rise to NOE correlations!

• 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

C

H

C

H5Å

NOE1 Å = 1x10-10 m

B Volkman - MCW

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, α-helix, β-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, α-helix, β-sheet).

NMR

Sample preparationHow do we prepare the sample ?

∼ 0.5 ml of solution

Concentration ca. 1mM

5 mm

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)

NMR

Sample preparation - the deuterium lockMagnets 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

(to reduce aggregation)

(to control pH)

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

Sample preparationW Westler - UMadison

NMR

Sample preparationW Westler - UMadison

NMR

Sample amount

For 400µl 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 exchangeIn 100% D2O: !!Amino acids and small peptides - all NHs exchange to NDs. !Peptides and proteins - 1º backbone NH exchanges fast, the rest eventually exchange. !!In 9/1 H2O/D2O: !Proteins all backbone NHs except 1º seen Amino acids and peptides (low pH) 1º NH can appear

NMR

pH and chemical exchange

Fast H2O↔ NH exchange = loss of peak intensity

Slow H2O↔ NH exchange = no 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 sequencesPlace 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

NMR Measurables Information Obtainedprecessional 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: φ, ψ

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

Information from NMR experiments

NMR

• Scalar ( J ) coupling: arises between nuclear spins separated by a small number of covalent bonds : COSY, TOCSY, HMQC, HSQC

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

C

H

C

H5Å

NOE

C C H

H

H

H J

Two basic types of internuclear correlationsB Volkman - MCW

NMR

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: !!!!!

•Each spin now has two energy ‘sub-levels’ depending on the state of the spin it is coupled to: !!!!!!!• 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.

1 3 C

1 H 1 H 1 H

one-bondthree-bond

αα

αβ βα

ββ

I SS

S

I

IJ (Hz)

B Volkman - MCW

NMR

Constantes de acoplamentoSistema de 1ª ordem - &, >> J

CH3-C-O-CH2-CH3

O

# 1,5 ppm# 4,5 ppm J # 7 Hz

cada 1H no grupo CH2 “vê” quatro estados possíveis do CH3

cada 1H no grupo CH3 “vê” três estados possíveis do CH2

$$

%%

$%%$

CH3CH2

$$$

%%%

$$% $%$ $$%

$%%%$%%%$

# 1,5 ppm# 4,5 ppm

J # 7 Hz

1:3:3:1 1:2:1

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

NMR

# 1,5 ppm# 4,5 ppm

Constantes de acoplamentoSistema de 1ª ordem - &, >> J

CH3-C-O-CH2-CH3

O

# 1,5 ppm# 4,5 ppm J # 7 Hz

cada 1H no grupo CH2 “vê” quatro estados possíveis do CH3

cada 1H no grupo CH3 “vê” três estados possíveis do CH2

$$

%%

$%%$

CH3CH2

$$$

%%%

$$% $%$ $$%

$%%%$%%%$

# 1,5 ppm# 4,5 ppm

J # 7 Hz

1:3:3:1 1:2:1

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

amino acid/small peptide protein

NMR

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 Å

saturate

dec on

off – on

dec off

NOE

• 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

B Volkman - MCW

NMR

1D Spectra for Amino acids

NMR

1D NMR1D 1H spectrum of Alanine in 100% D2O

CH3

CH

TSPHODH

NMR

1D NMR

H

CH3 - 1:1

CH - 1:3:3:1

Area 1 Area 3

1D 1H spectrum of Alanine in 100% D2O

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

2D NOESY

t1 τM

1D Fourier Transform

2D Fourier Transform

How do we avoid overlap ?

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 experiências (J.Chem.Phys. 1975)

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

Métodos baseados no acoplamento de dipolos nucleares: interacções podem ser escalares (através das ligações) ou dipolares (através do espaço).!

transferência de magnetização por permuta química

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

- HETERONUCLEARES 1H-15N/13C: HSQC / HMQC

NMR

2D NMR - experiment

Preparation - Waiting time for system to

return to equilibrium - Followed by one or more

pulses Evolution - Evolution spin systems are not in

equilibrium - Varies expt to expt.

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

Detection - Delay during which the signal is

acquired (t2)

Preparation Detection

1D Delay then pulse then acquire

Preparation MixingEvolution Detection

t1 t2delay,pulsedelay,pulse

2D Delay then pulse then incremented delay then mixing then acquire

2D = all correlations can be seen in one expt.

NMR

x

FT(t1)

z

x

t1

90ºx 90ºx

FT(t2)

Modulação da amplitude do sinal variando t1

x

x

2D NMR - acquisition and processing

NMR

2D NMR - acquisition and processing

NMR

2D NMR

A BONUS→regions in 2D spectra provide protein fingerprints

If 2D cross peaks overlap→ go to 3D or 4D

NMR

Homonuclear 2D experiments

COSY

NOESY

TOCSY

- Dão origem a espectros caracterizados por uma diagonal (com simetria relativamente à diagonal)

- O tempo de mistura é desenhado para a selecção de cada tipo de interacção entre núcleos: transferência de magnetização baseada em acoplamento escalar ou interacção dipolar

J

espacial

J,J

MP DE

spinlock

tmixt1

t1

NMR

COSY - correlation spectroscopy

t1 t2

F1

F2

Núcleos A e X (JAX)

t1d1

Permite obter informação acerca das constantes de acoplamento

Perda de sensibilidade compensada com a clarificação da região diagonal

Utilizado quando queremos supressão de água.

Double Quantum Filtered COSY

NMR

COSY spectrum

i-1 i i+1

regiões bem definidas

aumento resolução relativamente a 1D

NMR

COSY correlations

ppm 0

HN Hα Hβ

10

Valina (V)

H3C

N C C

O

H

β CH

H

CH3γ

α

ppm

ppm

HN Hα Hβ Hγ

10 0ppm 010

Correlações 3J - 3 ligações químicas só...

C

O

Cisteína (C)

HS

N C C

O

H

β CH2

H

αC

H

α N

H

NMR

COSY spectrum

i-1 i i+1

Para identificar os desvio quimicos todos de um amino acido temos que andar em zonas com sobreposição

Amino acid 1

Proton ppm

NH 9.25

Hα 3.36

Hβ 1.57

Hγ 0.58

NMR

TOCSY - total correlation spectroscopy

Os vectores estão ‘locked’ ao longo do eixo B1

B1

- A sequência de mistura permite vários ‘saltos’ da magnetização, espalhando-a por um grupo de protões interligados por uma constante de acoplamento J.

- transferência de magnetização baseada em acoplamento escalar

Pulso 180º refoca a evolução dos desvios químicos; acoplamentos spin-spin mantêm-se activos

tmix

spinlockt1

CH2-CH-CH-O-CH-CH2

H H H H H

X

NMR

TOCSY correlations

Valina (V)

H3C

N C C

O

H

β CH

H

CH3γ

α

ppm0

Hα Hβ

10

ppm

HN

Hα

Hβ

Hγ

ppm 010

C

O

Cisteína (C)

HS

N C C

O

H

β CH2

H

αC

H

α N

H HN

NMR

2D NMR2D TOCSY spectra of Alanine in 100% D2O

NMR

2D NMR2D TOCSY spectra of Alanine2D TOCSY spectra of Alanine in 100% D2O

NMR

2D NMR2D TOCSY spectra of Alanine in 10% D2O

NMR

TOCSY patternsEach 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 patternsEach 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 78

NMR

TOCSY patterns

97

8

Hα

HβHγHδ

Hε

HN

NMR

TOCSY, COSY comparison

NOESY TOCSY COSY

NMR

tmixt1

ANOE α 1/rij6

Transferência de magnetização através de acoplamento dipolar

-informação 3D

-tmix

N

HH3C

H2CO

NH2H

H

NH3+

COO-

G10A25

N64

G125

Informação acerca de núcleos d < 5Å

NOESY

NMR

NOESY, TOCSY, COSY comparison

NOESY TOCSY COSY

NMR

NMR resonance assignment strategies!Atribuição específica das ressonâncias; correlação entre os picos do espectro de RMN e todos os 1Hs da proteína

!!!!

Definição das restrições conformacionais; distâncias entre protões e ângulos de torsão. Determinação de elementos da estrutura secundária

!

Cálculo da estrutura terceária

NH S35 HA Q135Estratégias para a atribuição das ressonâncias

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 strategiesStrategy 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 1Proteínas < 15kDa

- Sem marcação isotópica: até 10kDa

!1- Identificação das ressonâncias de cada amino ácido (sistema de spin)

!!2- Atribuição sequencial e específica

!!!Utilização dos espectros TOCSY (COSY) e NOESY

LTG S S R G

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

NMR

Assignment strategy 1

2D TOCSY: intra-residue correlations between protons

2D NOESY: inter-residue correlations between protons

ii-1 i+1

NMR

Assignment strategy 1

2D TOCSY: intra-residue correlations between protons

2D NOESY: inter-residue correlations between protons

ii-1 i+1

NMR

Sequential assignment

NMR

Chemical shifts

Approximate Chemical Shift Values for 1H’s in Different

Amino Acid Types��

“ballpark” starting values:�NH: 6-10 ppm�Hα: 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 chemical shifts

NMR

Chemical shifts - BMRB database

NMR

Chemical shifts - BMRB standard compounds

NMR


NMR


NMR

Chemical shifts - BMRB alanine spectrum

NMR

Chemical shifts - BMRB update display

NMR

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

Hα: 5.1 ppm

Hβ: 1.9 ppm

Hγ,γ’: 0.9 ppm

These NMR peaks are in the same residue, and the

residue type is Val

Typical peak pattern for Val in a TOCSY spectrum

Conclusion we have a valine that has 1H chemical shifts of 8.1(HN), 5.1(Hα), 1.9(Hβ) and 0.9(Hγ) ppm - BUT which valine is it in the sequence ??

NMR


N CH C

R2

O

CH C

R1

O

N

H H i+1iX

TOCSY - definem-se as correlações entre todos os protões de um amino ácido - identificam-se os resíduos por padrões característicos

AMX

Correlações dentro do mesmo resíduo aa

NMR


2D TOCSY: correlações entre protões intra-resíduo 2D NOESY: correlações entre protões inter-resíduo

NOESY!- atribuição sequencial dos aas: correlações entre aas consecutivos (e dentro do mesmo aa)!- atribuição específica (é necessário conhecer a sequência primária)

1 2 3 4 5 6 7 !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


NMR

Strategy 1 for establishing assignments

1° sequence

The combination of the NMR results with the known primary sequence gives the resonance 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 it’s a T then we know the assignment is T19-V20-S21 - if it’s a H then we have H29-V30-S29 etc…..

NMR

Strategy 1 for establishing assignments2D TOCSY

through-bond connections (≤ 3 bonds)

intra-residue connections

defines residue type

2D NOESY through-space

connections (≤ 5Å)

inter-residue connections

defines residue neighbors

known 1° sequence

!Sequence-specific resonance assignments

Clusters of NMR peaks consistent with peptide segments

+

NMR

1H resonance assignment table

the end result of Stage I (strategy 1)

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

NMR Measurable !1. 1H-1H NOE

!!

2. chemical shifts !!!3. 3J coupling

constants

Information Obtained !1. interproton distances (<5Å) !2. backbone dihedral

angles, secondary structure

!3. dihedral angles

Stage II - conformational restraints

NMR

Diferenças dos desvios químicos dos protões HA, relativamente aos valores ‘random coil’!!Valores > RC (código= +1) Folha beta!!Valores < RC (código= -1) Hélice alfa

Chemical shift index - CSI (Hα)

NMR

13C spectrum of a protein

NMR

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

Hélice α Folha β

!Cα, CO

!+1

!-1

!Cβ

!-1

!+1

!Hα

!-1

!+1

Chemical shift index - CSI (Cα)

NMR

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 -1’s not interrupted by a 1 is alpha-helix. Any “dense” grouping of three or more 1’s not interrupted by a -1 is a beta-strand. All other regions are designated as coil.!!(4) A minimum of three consecutive 1‘s is needed to define a beta-strand,and a minimum of four (not necessarily consecutive) -1‘s 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 zero-valued chemical shift indices marks the termination point.

Chemical shift index - CSI - Procedure Hα

NMR

Chemical shift index - CSI - Procedure CαNeed 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 chemical-shift indices for Ca and carbonyl carbons, one may identify the secondary structures as follows:!!(3) Any 'dense' grouping of four or more 1’s not interrupted by a -1 is a helix. Any dense grouping of three or more -1‘s not interrupted by a 1 is a b-strand. All other regions are designated as coil.!!(4) A minimum of three consecutive -1‘s is needed to define a b-strand, and a minimum of four 1‘s 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

AROM

13C

J-couplings in proteins

NMR

1H-13C HSQC

1H

13C

NMR

1H-13C HSQC

NMR

Sequential and Medium-range NOEs identify α-helices

• Predictable NOE patterns (short inter-proton 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

2ª structure from NOE patterns

- Podem ser identificados através dos padrões de NOE

NMR


NMR


i

i+3

i+4

NMR

Karplus relation - peptide torsion anglesMartin 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:

A, B, and C are empirically derived parameters.

J couplings provide a semi-quantitative measure of molecular conformation

J(θ)

θ

θ

NMR

Karplus relation - peptide torsion angles

NMR

3D structure from NOEs

N

HCa

H5Å

NOE

Met 12 Pro 21

C

N

Brian Volkman - MCW

NMR


N

HH3C

H2CO

NH2H

H

NH3+

COO-

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

G10A25

N64

G125

NMR


N

HH3C

H2CO

NH2H

H

NH3+

COO-

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.

G10A25

N64

G125

NH G10 – CH3 A25 4.0 Å

CH3 A25 – CH2 N64 4.5 Å

CH2 N64 – HA G125 3.0 Å

NH G10 – HA G125 2.5 Å

ANOE = C. 1/(rij6)

NMR

t1

NOESYtmix

Transferência de magnetização através de acoplamento dipolar

NH G10 – CH3 A25 4.0 Å

CH3 A25 – CH2 N64 4.5 Å

CH2 N64 – HA G125 3.0 Å

NH G10 – HA G125 2.5 Å

N

HH3C

H2CO

NH2H

H

NH3+

COO-

G10A25

N64

G125

Informação acerca de núcleos d < 5Å

Integração de todos os picos: !programa SPARKY (XEASY, CARA)

Strong NOE 1.8 - 2.7 Å Medium NOE 1.8 - 3.3 Å Weak NOE 1.8 - 5.0 Å

ANOE = C. 1/(rij6)

NMR

NOESYInformação acerca de núcleos d < 5Å

46 LYS ! HN 46 LYS HB2 3.43 ! HN 46 LYS HB3 3.37 ! HN 46 LYS QG 5.51 ! HN 46 LYS QD 6.88 ! HN 47 SER HN 3.42 ! HA 46 LYS HB3 3.27 ! HA 46 LYS QD 6.88 ! HA 48 GLU HN 3.95 ! HA 49 PHE HN 4.29 ! HA 49 PHE QD 6.10 ! HB2 46 LYS QE 3.81 ! HB2 46 LYS QZ 5.82 ! HB2 47 SER HN 4.20 ! HB3 46 LYS QE 6.72 ! HB3 46 LYS QZ 4.99 ! QG 46 LYS QZ 7.32 ! 47 SER ! HN 47 SER HB2 3.27 ! HN 48 GLU HN 3.18 ! 48 GLU ! HN 48 GLU HB2 3.11 ! HN 48 GLU HB3 3.58 ! HN 48 GLU HG2 4.48 ! HN 48 GLU HG3 3.70 ! HN 49 PHE HN 2.80 ! HN 49 PHE QD 6.66 ! HA 48 GLU HB3 2.96 ! HA 48 GLU HG3 3.21 ! HA 49 PHE HN 3.33 ! HB2 49 PHE QD 7.62 ! 49 PHE ! HN 49 PHE HB2 3.55 ! HN 49 PHE HB3 3.30 ! HN 50 GLU HN 4.17 ! HA 50 GLU HN 2.40 ! HB3 50 GLU HN 3.39 ! QD 50 GLU HN 6.35 !

NMR


Stage I: Establish sequence-specific resonance assignments

!!



!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 φ,ψ 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

- cadeia polipeptídica numa conformação ao acaso

- Utilização de métodos de mecânica clássica de minimização da energia total do sistema – e.g. ‘Simulated Annealing’. Incorpora as restrições experimentais numa função de energia

Simulação do movimento dos átomos em condições de aquecimento. A proteína é ‘aquecida’ provocando movimentos moleculares. Depois é arrefecida lentamente, de maneira a minimizar a energia.

r R

Energy

Programas que utilizam diferentes métodos para calcular uma família de estruturas (confórmeros) : XPLOR, DYANA, CYANA

NMR

Structure determination - the problem a mathematical calculation that converts a table of

distances into a map or structure

Input: intercity distance table Output: map of US

For NMR data: Input: Output: 1H - 1H distance table structure of molecule

46 LYS ! HN 46 LYS HB2 3.43 ! HN 46 LYS HB3 3.37 ! HN 46 LYS QG 5.51 ! HN 46 LYS QD 6.88 ! HN 47 SER HN 3.42 ! HA 46 LYS HB3 3.27 ! HA 46 LYS QD 6.88 ! HA 48 GLU HN 3.95 ! HA 49 PHE HN 4.29 ! HA 49 PHE QD 6.10 ! HB2 46 LYS QE 3.81 ! HB2 46 LYS QZ 5.82 ! HB2 47 SER HN 4.20 ! HB3 46 LYS QE 6.72 ! HB3 46 LYS QZ 4.99 ! QG 46 LYS QZ 7.32 ! 47 SER ! HN 47 SER HB2 3.27 ! HN 48 GLU HN 3.18 ! 48 GLU ! HN 48 GLU HB2 3.11 ! HN 48 GLU HB3 3.58 ! HN 48 GLU HG2 4.48 ! HN 48 GLU HG3 3.70 ! HN 49 PHE HN 2.80 ! HN 49 PHE QD 6.66 ! HA 48 GLU HB3 2.96 ! HA 48 GLU HG3 3.21 ! HA 49 PHE HN 3.33 ! HB2 49 PHE QD 7.62 ! 49 PHE ! HN 49 PHE HB2 3.55 ! HN 49 PHE HB3 3.30 ! HN 50 GLU HN 4.17 ! HA 50 GLU HN 2.40 ! HB3 50 GLU HN 3.39 ! QD 50 GLU HN 6.35 ! 50 GLU ! HN 50 GLU HB2 3.52 ! HN 50 GLU HB3 4.07 ! HN 50 GLU HG2 3.76 ! HN 50 GLU HG3 3.98 ! HN 51 ALA HN 4.48 ! HA 51 ALA HN 2.74 ! HG2 51 ALA HN 4.20 ! HG3 51 ALA HN 3.30

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

+

NMR

Cyana structure calculation

Note: many structures have similar final target function energies!!In NMR we always have a family of final structures

NMR

Cyana structure calculation

30 TAD structures !w/disulfide bond only

30 TAD structures !w/disulfide and !NOE restraints

Assign NOEs, generate !distance constraints

Calculate 60 structures with !

TAD (DYANA)

Brian Volkman - MCW

NMR

Distance restraints

experimental distance constraint

NMR

Distance restraintsRestrições de distâncias experimentais

Numero de restrições: definição da estrutura

A – 322 NOEs

B – 657

C – 747

D – 809

Conclusão: quanto mais restrições experimentais melhor!

NMR

Distance restraintsPara aumentar o nº de restrições (NOEs) podemos: !1. Aumentar a concentração da

amostra 2. Aumentar o nº de scans na

experiencia NOESY 3. Fazer o espectro a um campo

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

NMR

NMR familyCadeia principal

Cadeias laterais

Estrutura de RMN representada por um grupo de estruturas sobrepostas (10-30)

Zonas indefinidas devidas a

- Falta de restrições

ou

- Flexibilidade Molecular Determinação de parâmetros de relaxação (T1 ou T2), permite detectar mobilidade de cada resíduo

RMSD (root mean square deviation) entre os vários modelos serve para determinar a convergência no cálculo das estruturas (precisão) ! N

R = √ (1/N) ∑ (ri – ri’)2 i=1

Boas estruturas: RMSD(BB)< 1 Å

NMR

NMR familyZonas indefinidas devidas a

- Falta de restrições

ou

- Flexibilidade Molecular

Determinação de parâmetros de relaxação (T1 ou T2), permite detectar mobilidade de cada resíduo

NMR

Structure visualisationRibbon

Sobreposição indentifica zonas pouco definidas

Com todas as ligações

Só cadeia principal

Sobreposição de 15 estruturas

NMR

Structure determination by NMR

1970 1980 1990 2000

RMN transformada de Fourier

NOESY, TOCSY

RMN 2D

Atribuição proteinas 1H/ estruturas 3D

Ressonância Tripla

Atribuição de proteínas 13C/15N

RDCs

TROSY

Métodos de aquisição rápida

Detecção 13C para Bio-RMN

Biologia Molecular

Proteínas recombinantes, expressão e marcação isotópica

60aa 160aa 260aaTamanho da proteína

NMR


NMR


Backbone conformation from chemical shifts (Chemical Shift Index- CSI): ψ,φ !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


Stage I: Establish sequence-specific resonance assignments

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Stage III: Calculate and refine the 3° structure

!YOU ARE HERE!

NMR

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

Assessing the quality of NMR structures

NMR

NMR v X-ray CrystallographyAdvantages 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 versus X-rayThe 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

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!

NMR versus X-ray

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