A one-dimensional (1D) NMR spectrum of a protein

CO N

HΝ

Cα CO

Hα

HN Hα

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Ω(HN)

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Ω(Hα )

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Ωref

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δ ( )ppm=(Ω− Ωref )/Ωref

Chemical shifts in parts per million (ppm)Are independent of the field strength of theStatic magnetic Bo field.

See the supplementary lecture material andRattle, ‘NMR Primer for Life ScientistsPages 19-21, 26.

9

Backbone HN

Aromatics

H2O

Hα

Upfield shiftedmethyls

Methyl

8 7 6 5 4 3 2 1 0

1H chemical shift (ppm)

600.134800 600.132400600.130000

For a 1H protein spectrum we need a fair amount of protein, maybe 5-10 mgs of protein. 600 L sample volume - gives a 1-2 mM concentration of protein.

The 1D 1H spectrum of a protein

0123456789ppm

Amides

Aromatics

H2O

CαH

CH

CH CδH

Upfield shiftedmethyl

A lot of work done with histidine since the C2 proton appears at higherfrequency than most other protons.

C

H

COO-+H3N

C

C

NC

N

C

HH

H

+1

2

3

4

H H

H6.8-7.2 ppm

8.0-8.8 ppm

1H NMR spectrum of Histidine - C2 proton appears at higher frequency than most other protons and is sensitive to the protonation of the ring.

C2H C4H CαH CH

010

Raise pH

10 0ppm

8.0-8.8 ppm

6.8-7.2 ppm

C

H

COO-+H3N

C

C

NC

N

C

HH

H

+1

2

3

4

H H

H

Shown in protonated form

α

pH1 3 5 7 9 11

ChemicalShiftChangeδ ppm)

0

1

Titration of the C2H of Histidine

pKa = 5.2

Shift measured with multiple 1D spectra starting withpH 1.0 and moving through to pH 9

The chemical shift change of the proton on C2 reflects the protonation of N1

50% ofcomplete change

C

H

COO-+H3N

C

C

NC

N

C

HH

H

+1

2

3

4

H H

H

4 histidines which couldbe monitored and have their pKa’s measured.

H1 = His105H2 = His119H3 = His12H4 = His48

Measure pKa of each histidine

pKa

His105 6.7

His119 6.2

His12 5.8

His48 is more complex,sudden discontinuity in thecurve.

Found that 200mM Na+CH3COO-

helped to stabilize the protein.

Can then determine that the pKaof C2H is 6.31.

There is a conformational change affecting this peak so that at some pHs two peaks were observed. H4a and H4b were acid and basestable forms.

His105

His12

His48

His119His48 and His105 are unchanged

His12 and His119 curved are shifteddownfield.

Why downfield??

His119 changes from 6.2 to 8.0His 12 changes from 5.8 to 7.4

Both His12 and His119 are protonated in the enzyme-inhibitor complex. The proton is protected from exchange by the presence of the inhibitor. Need to go to higher pH to remove it.

Repeat titrations in the presenceof an inhibitor.

in this case, cytidine-3’-monophosphate (3’-CMP) O

OPO3- OH

NHOCH2

NH2

O

O

OPO3- OH

NHOCH2

NH2

O

A simple 1D NMR Spectrum - this is ethanol but the spectrum has the sort of simplicity we might get with a one amino acid protein(there is no such thing!)

A 36 amino acid proteinA successful NMR experiment comes in 3 stages,

1.) Resolve the resonances - that is, obtain a spectrumwhere individual signals are clearly resolved from oneanother.

2.) Assign the resonances. Each peak comes from oneatom in the protein - but which one? Our 36 amino acidprotein is a mess! The record to date is 723 amino acidsWith full assignment of the spectrum - how did they dothis?

3.) Interpret the data.

Effect of increasing spectrometer frequency

1 GHz soon??

13

rad s-1

rad s-1 T-1. T

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ωo

2π= vo

vo =γBo

2π

s-1 (Hz)

Larmor Frequency

14

A compass in a magnetic field

15

A nuclear spin precesses in a magnetic field

the circulating motion of the spin angular momentum is called precession

Nuclear spins precess because:• they are magnetic•they have angular momentum

this arrow denotes the direction of the spin angular momentum

16

Precession frequency = Larmor frequency0 = - Bo/2π

Larmor frequency in Hz (= cycles per second)

gyromagnetic ratio in rad s–1 T–1

magnetic field inTesla (T)

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E = γhBo

ΔE = hωo

ωo = γBo

Compare with Zeeman Splitting

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,Since ωo = 2πvo

ωo = −γBo

x

y

zBo

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ωo = −γBo

α

Magnetic vectors precess around the static magnetic field at thelarmor frequency. There is a slight population excess in the low energy (α) state. This leads to a net magnetization along Z (in green). There is no net magnetisationalong x or y as this is essentially randomised.

x

y

α

x

y

α

Generation of transverse magnetization by π/2 pulse

x

y

z

(/2)x

x

y

z

/2

Magnetization perpendicular to the magnetic field is called transverse

magnetization

yNet Moment

NMR signal

Note the orientation of the coil -perpendicular to the magnetic field x

y

z Bo

Preamp

The NMR signal is also called the free induction decay

(fid)

Precession of Transverse Magnetization

The transverse magnetization components oscillate and decay

Mx

My

Time

Time

x

y

z

x

y

z

x

y

zBo

xy plane

My (t) = -Mzeq cos(ωt) exp{-t / T2}

Mx (t) = Mzeq sin(ωt) exp{-t / T2}

oscillation at the Larmor frequency

decay time constant = spin-spin relaxation time OR transverse relaxation time

Pg 46 & 47 of Rattle

Diagonal

DiagonalCrosspeaks

COSY of Alanine in D2O

COSY Spectrum of a small protein

Areas of Spectrum

Typical Amino Acid spin-system patterns on COSY spectra

1.) Just see 3Jcoupling

2.) Do notsee couplingsacross the peptide bond.