39
Chapter I
For quantification relative peak intensities were measured and compared (figure
2.10). Six peaks showed significant changes in peak intensity. Four stronger peaks were
affected as well as two weaker peaks. Interestingly all six peaks showed an increase in
intensity after MLE addition.
Figure 2.10: Comparison of relative peak intensities of tr-HSQC spectra of
15
N,
13
C,
2
H-MBP
peptide in complex with DR2 before (blue) and after (red) addition of J10-1. Peaks of the tr-HSQC
spectra were numbered and relative intensities determined. Blue diamonds indicate peaks of the spectrum
without MLE addition. Red squares represent peaks of the spectrum after MLE addition. Significant
intensity changes are indicated with black circles.
2.3.7
Performing HNCA and
15
N-NOESY experiments of HLA-DR2/
15
N,
13
C,
2
H-
MBP to determine resonance assignments
To further investigate which parts of the peptide might have been affected
by binding
of the small molecule J10-1, it was necessary to assign the resonances to specific
peptide residues, especially the ones showing a change in intensity. Therefore, using the
same DR2/
15
N,
13
C,
2
H-MBP sample which was used for measuring the HSQC spectrum
of the complex, HNCA and
15
N-NOESY experiments were carried out. By matching C
α
chemical shifts obtained from the HNCA experiment and additional spatial information
from the
15
N-NOESY experiment some sequential assignments were made (see table
2.1). But unfortunately the resonance assignments were not unambiguous which can be
also seen in table 2.1. As there were more peaks than expected for a 15 amino acid long
peptide and apparently several distinct peptide conformations present, it was not clear
how many peaks actually arose from each peptide residue and which peaks belonged to
a certain peptide conformation. Only some C
α
chemical shifts could be matched to C
α
chemical shifts of neighboring peptide residues and it was not possible to determine a
complete C
α
connectivity. Without knowing the C
α
connectivity it was also not possible