Download Lecture2-2010

A one-dimensional (1D) NMR spectrum of a protein
H
HN
H
H 2O
H
CO N
Methyl
C CO
Backbone HN
H
Aromatics
(H N )
(H )
ref
600134800 600132400 600130000


600.130000
(Hz)
(MHz)

 (ppm) (ref ) / ref
9
8
1H

7
6
5
4
chemical shift (ppm)
3
2
1
0
Upfield shifted
methyls
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.
1
Y215Q
2
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
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.
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??
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.