68
Chapter II
3.3.8
Measuring HLA-DM binding to HLA-DR1 carrying an HA peptide variant
with a valine at P1 position and two N-terminal peptide residues absent
As the DR1/peptide complex used for SPR (figure 3.3) and crystallization
experiments (figure 3.4) contained two HA peptide variants, one missing a P1 residue
and the other one containing a valine at P1 position, shown by mass spectrometry
(figure 3.11), it had to be determined whether both or only one of the complexes bind to
DM. Previous experiments (Anders et al., 2011) had shown that if the DR1 molecule
carried an HA peptide variant missing two N-terminal residues and contained a tyrosine
at P1 position, no DM binding was observed. The question was whether DM binding
could be detected with a DR1/peptide complex including a smaller anchor residue as
valine. Therefore, DR1/peptide complexes were prepared with synthesized HA peptide
variants missing residues P-2, P-1 and one peptide specifically containing a valine at P1
position and another one containing a glycine at P2 position and no P1 residue. Both
complexes were used to carry out SPR experiments. The HA peptide with a glycine at
position P2 (P-2, P-1, P1 missing) was synthesized by a different company and highly
purified after synthesis to avoid contaminations. A glycine was chosen for the P2
position to ensure that even if a by-product of the peptide synthesis would include an
additional glycine at position P1 it would not represent an anchor residue.
As can be seen in figure 3.13 DM binding was observed for the DR1 complex
carrying an HA peptide missing two N-terminal peptide residues and containing a
valine at P1 position although significantly less than for the previously used DR1
complex containing a mixture of HA(P
1,Val
-P
11
) and HA(P
2
-P
11
) peptides. More DM
binding than to the previously used DR1 complex was observed with the DR1/peptide
complex carrying an HA peptide with a glycine at P2 position and most probably no P1
residue. In figure 3.13 (B) it can be seen that DM binding to the DR1/complex
containing an HA peptide with a valine at P1 position (HA(P
1,Val
-P
11
)) is concentration
dependent. Interestingly, the off-rate of DR1/HA(P
1,Val
-P
11
) is slower compared to the
off-rates of DR1/HA(P
2
-P
11
) and DR1/HA(P
2,Gly
-P
11
) which could indicate that the
valine at P1 position may stabilize a DR conformer that binds to DM.