40
Chapter I
to assign the chemical shifts to specific peptide residues. Due to this complexity
unfortunately it could not be determined which peptide residues might have showed an
increase in signal intensity upon small molecule addition.
Table 2.1: Possible peak assignments based on HNCA and
15
N-NOESY experiments. The first
line displays the peptide sequence. Rows below show peak numbers with possible assignments to specific
peptide residues. Numbers in parenthesis show C
α
chemical shifts in ppm. Numbers with matching color
indicate sequences of identical peaks which can be assigned to different peptide residues. Peak numbers
refer to peaks indicated in the tr-HSQC spectrum in figure 2.9 (A).
E N
S
V
V
H
F
F
K
N
I
V
T
S
R
36
(58)
24
(61)
60
(63)
8
(53)
33
(55)
34
(58)
27
(62)
34
(58)
27
(62)
23
(62)
25
(54)
18
(62)
3
(56)
23
(62)
25
(54)
18
(62)
3
(56)
9
(55)
5
(59)
9
(55)
5
(59)
7
(49)
2.3.8
Measuring
19
F-NMR spectra of J10-11 in the presence of HLA-DR2/MBP
and HLA-DR2/CLIP
To further investigate the binding of J10 and its derivatives to DR2 one-dimensional
19
F-NMR spectra of the small molecule were measured in the presence of DR2/peptide
complexes with different affinities. Some of the J10 derivatives include fluorine atoms
which possess NMR active nuclei.
19
F atoms are naturally abundant and have a nucleus
with spin of ½, same as protons. As nuclei with spin of ½ undergo splitting of spin
states in an external magnetic field the nuclei can be excited by electromagnetic
radiation yielding measurable NMR signals.
The advantage of
measuring
19
F-NMR
spectra instead of
1
H-NMR
spectra is that
19
F-
NMR signals can only derive from the small molecule as the protein contains no
fluorines which simplifies the spectra. A one-dimensional
1
H-NMR spectrum would be
42
Chapter I
Figure 2.11: Comparison of three different one-dimensional
19
F-NMR spectra of the MHC II
loading enhancer J10-11 free in solution (grey), in the presence of DR2/MBP (blue) and in the
presence of DR2/CLIP (green). The spectra were collected with a small molecule concentration of 0.25
mM in 50 mM citrate buffer (pH 5.2) at 25 ºC using a NMR machine with a magnetic field strength of
11.7 Tesla. The five peaks of the three different spectra are overlapping but for comparison the spectra of
J10-11 in the presence of 11 μM DR2/MBP (blue) and 11 μM DR2/CLIP (green) were shifted to the
right. The signal on the right refers to 1 mM TFA which was added to the NMR tube outside of the
microcapillary. The three NMR spectra were compared by matching the peak height of the TFA standard
and the noise level. Numbers next to peaks and next to fluorine atoms of the small molecule J10-11
displayed in the top left show the peak assignment.
2.4
Conclusion
To further improve the peptide loading activity of the small molecule J10, which has
a potential as adjuvant for peptide therapeutics, a better understanding of the exchange
mechanism is necessary. Two different methods were applied to gain a comprehensive
understanding of the small molecule interactions with the MHC II/peptide complex and
their impact
on peptide-binding, i.e. X-ray crystallography and NMR spectroscopy.
First, DR2/MBP was crystallized in the presence of the J10 derivatives J10-1 and
J10-12 and the crystals diffracted till 2.9 Å. A data set from a co-crystallization trial
showed extra electron density close to the peptide N-terminus in the region around
residue Ser α53. Previous experiments with covalent linkage of the small molecule to
the peptide had shown that the small molecule binds in an area close to the peptide N-
terminus (Call et al., 2009). Also, mutagenesis studies of the α chain
close to the peptide