34
Chapter I
Thirteen instead of seven peaks were observed that were likely arising from peptide
backbone,
three stronger, six intermediate and four weaker peaks.
But as some peak intensities were still low and therefore peak changes upon small
molecule addition might have been difficult to detect it was decided to further enhance
the signal by increasing the concentration of the protein complex and by deuterating the
peptide besides
15
N- and
13
C-labeling. The advantage and effects of deuterating the
peptide will be explained in the next section.
Figure 2.6: tr-HSQC spectrum of
15
N-labeled MBP peptide in complex with DR2 measured with
a
1
H frequency of 900 MHz. The spectrum of DR2/
15
N-MBP was measured in 10 mM Tris (pH 5.2) and
100 mM NaCl at a concentration of 0.35 mM at 37 ºC. The
1
H frequency was 900 MHz and 256 scans
were measured.
35
Chapter I
2.3.5
Deuterating MBP peptide to improve signal intensity of the HSQC
spectrum of isotope labeled MBP peptide in complex with HLA-DR2
As described in the previous section the detected NMR signal resulting from
15
N-
labeled MBP peptide in complex with DR2 was very weak. As
the peptide was bound to
a large protein (~ 47 kDa) its molecular tumbling rate corresponded to the molecular
tumbling rate of the higher molecular weight complex leading to signal loss. To
improve signal intensity temperature, magnetic field strength and scan number were
increased. But still not all expected resonances from the peptide were detected. Another
possibility to enhance signal intensity arising from backbone amide protons is to
minimize spin-spin interactions with neighboring protons by substituting non-
exchangeable protons with deuteriums.
As the MBP peptide binds to the DR2 molecule in an extended conformation the
backbone amide protons are in close proximity to protons of the C
ß
-atom of the
neighboring residue causing fast relaxation and decrease in signal strength. The same
applies to the C
α
-proton of the same residue which is next to the amide proton of the
peptide backbone. This effect of fast relaxation can be eliminated by replacing the non-
exchangeable
protons of the C
α
- and C
ß
-atoms with deuteriums.
Another factor for signal loss is the close contact of the MBP peptide with DR2
residues. Again through spin-spin interactions of the resonating protons of the peptide
with resonating protons of the MHCII molecule the signal of the peptide is further
depleted. But as the DR2 protein is expressed in CHO cells which is a complex
expression system a new protocol in a different expression system would have had to be
established. Therefore only the peptide was deuterated. Furthermore, the MBP peptide
was
13
C-labeled to allow triple resonance experiments
determining C
α
-connectivity.
As bacterial growth in D
2
O is very slow compared to H
2
O the bacterial culture was
gradually adjusted
to media prepared with D
2
O. For
15
N- and
13
C-incorporation
15
NH
4
Cl
and
13
C-glucose was added to M9 minimal media. The purification protocol was the
same as for
15
N-labeled MBP peptide (figure 2.2). 3 mg of
15
N,
13
C,
2
H-MBP peptide
were prepared and the yield was 2.5 mg of triple-labeled MBP peptide per 1 liter
bacterial culture. Successful triple-labeling of the MBP peptide was confirmed by mass
spectroscopy (see figure 2.7).