Transcript (1D) NMR spectrum of a protein



A one-dimensional (1D) NMR spectrum of a protein
H
HN
H
CO N
H 2O
H
Methyl
C CO
Backbone HN
H
Aromatics
(H N )
(H )
ref
600.134800 600.132400 600.130000


 (ppm)  ( ref )/ref
Chemical shifts in parts per million (ppm)
Are independent of the field strength of the
Static magnetic Bo field.
See the supplementary lecture material and
Rattle, ‘NMR Primer for Life Scientists
Pages 19-21, 26.
9
1H
8
7
6
5
4
chemical shift (ppm)
3
2
1
0
Upfield shifted
methyls
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
H2O
CgH CH
CbH
Aromatics
CH
Amides
Upfield shifted
methyl
9
8
7
6
5
4
ppm
3
2
1
0
H
+H3N
C
COO-
H
C
H
+
4
C
H N
1
C
C2
H
N3 H
H
8.0-8.8 ppm
A lot of work done with histidine since the C2 proton appears at higher
frequency than most other protons.
6.8-7.2 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.
H

+H3N
C
H
+
COO
C
H N
1
C
C2
CbH
6.8-7.2 ppm
Cb H
4
CH
C2H C4H
-
H
0
10
N3 H
Raise pH
H
8.0-8.8 ppm
Shown in protonated form
10
ppm
0
Titration of the C2H of Histidine
Shift measured with multiple 1D spectra starting with
pH 1.0 and moving through to pH 9
H
+H3N
C
COO-
H
C
H
C
C
The chemical shift change of the proton on C2 reflects
the protonation of N1
+
H N
1
1
pKa = 5.2
0
1
3
C2
H
50% of
complete
change
Chemical
Shift
Change
D(ppm)
4
5
7
9
11 pH
H
N3 H
4 histidines which could
be monitored and have their
pKa’s measured.
H1 = His105
H2 = His119
H3 = His12
H4 = His48
Measure pKa of each histidine
pKa
His105
6.7
His119
6.2
His12
5.8
His48 is more complex,
sudden discontinuity in the
curve.
There is a conformational change affecting
this peak so that at some pHs two peaks were
observed. H4a and H4b were acid and base
stable forms.
Found that 200mM Na+CH3COOhelped to stabilize the protein.
Can then determine that the pKa
of C2H is 6.31.
Repeat titrations in the presence
of an inhibitor.
His105
in this case, cytidine-3’monophosphate (3’-CMP)
His48
O
N
O
OPO3-
His12
His119
HOCH2
NH2
OH
His48 and His105 are unchanged
His12 and His119 curved are shifted
downfield.
His119 changes from 6.2 to 8.0
His 12 changes from 5.8 to 7.4
Why downfield??
Both His12 and His119 are protonated in the enzymeinhibitor complex. The proton is protected from exchange
by the presence of the inhibitor. Need to go to higher pH
to remove it.
NH2
HOCH2
O
N
O
OPO3-
OH
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 protein
A successful NMR experiment comes in 3 stages,
1.) Resolve the resonances - that is, obtain a spectrum
where individual signals are clearly resolved from one
another.
2.) Assign the resonances. Each peak comes from one
atom in the protein - but which one? Our 36 amino acid
protein is a mess! The record to date is 723 amino acids
With full assignment of the spectrum - how did they do
this?
3.) Interpret the data.
Effect of increasing spectrometer frequency
1 GHz soon??
o
 vo
2
gBo
vo 
2 13
s-1 (Hz)
Larmor Frequency
rad s-1
.
rad s-1 T-1
T
A compass in a magnetic field
14
A nuclear spin precesses in a
magnetic field
the circulating motion of the spin angular
momentum is called precession
this arrow denotes the direction of the spin
angular momentum
Nuclear spins precess because:
• they are magnetic
•they have angular momentum
15
Precession frequency = Larmor
frequency
Larmor frequency in Hz
(= cycles per second)
gyromagnetic ratio in
rad s–1 T–1
n0 = - g Bo/2π
Since,  o  2v o
magnetic field in
Tesla (T)
 o  gBo
Compare with Zeeman Splitting

DE  g Bo
DE  o
o  gBo
16
Magnetic vectors precess around the static magnetic field at the
larmor frequency. There is a slight population excess in the low energy
() state. This leads to a net magnetization along Z (in green). There is no net magnetisation
along x or y as this is essentially randomised.
z
Bo



y
y
o  gBo
y
x

x
b
x
b
b
Generation of transverse magnetization by π/2 pulse
z
/2
z
y
(/2)x
x
y
x
Magnetization perpendicular to the
magnetic field is called transverse
magnetization
Net Moment
y
NMR signal
Bo
z
y
Note the orientation of the coil perpendicular to the magnetic field
x
Preamp
The NMR signal is also
called the free induction decay
(fid)
Precession of Transverse Magnetization
Bo
z
z
z
xy plane
y
y
x
x
Mx
y
x
oscillation at the Larmor frequency
Mx (t) = Mzeq sin(t) exp{-t / T2}
Time
decay time constant =
spin-spin relaxation time OR transverse relaxation time
My (t) = -Mzeq cos(t) exp{-t / T2}
Time
My
The transverse magnetization components
oscillate and decay
Pg 46 & 47 of Rattle
Crosspeaks
Diagonal
Diagonal
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 3J
coupling
2.) Do not
see couplings
across the
peptide bond.