65
Chapter II
3.3.6
Eluting N-terminally truncated HA peptide from HLA-DR1 protein used
for crystallization and analyzing by mass spectrometry
The peptide stock solution, used to prepare the crystallized DR1/peptide complex,
was analyzed to investigate whether a peptide contaminant derived from peptide
synthesis exhibited an additional valine at position P1 seen in the electron density
(3.3.5). Therefore, the peptide solution was analyzed by mass spectrometry in
combination with liquid chromatography and individual peptide sequences were
determined by identifying resulting peptide fragments. Indeed a minor peptide
component was found that included an additional valine at position P1
(VVKQNCLKLATK) besides the primary peptide with sequence VKQNCLKLATK.
To further investigate whether the peptide contaminant also bound to DR1, bound
peptide was eluted from DR1 protein used for crystallization. Under reducing
conditions the covalently linked peptide was released from DR1 and as the peptide
carried a C-terminal DNP-label the peptide was separated from protein using α-DNP
affinity chromatography. The eluted peptide sample was analyzed
by mass spectrometry
and the peptide contaminant was detected in addition to the primary peptide species. As
can be seen in the mass spectrum in figure 3.11, the particular peptide contaminant
carrying a valine at P1 position was even enriched on DR1 after peptide loading likely
due to the higher affinity to DR1 compared to the primary peptide missing a P1 anchor.
Therefore, DR1 protein used for crystallization contained both peptide species and
DR1 carrying the peptide contaminant including a P1 anchor might have crystallized
preferentially as it presumably exhibits higher stability.
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Chapter II
3.3.7
Measuring HLA-DR binding to N-terminally truncated HLA-DM using
surface plasmon resonance
As one of the two peptide-binding sites in the structure of DR1/HA(P
1,Val
-P
11
)
interacts with the flexible C-terminus of an adjacent DR1 molecule (3.3.5), the question
was raised whether the N-terminus of the DMα chain might bind to DR in
a similar way
and could be involved in peptide exchange. The N-terminus is present on the outer face
of DM and could reach the DM/DR binding site. Therefore, eleven residues of the DMα
chain were truncated and DR binding assays were performed using surface plasmon
resonance. However, as can be seen in figure 3.12, the truncated DM bound as well as
the unmodified DM molecule to DR1 carrying a low-affinity CLIP peptide. Therefore,
the possibility can be excluded that the N-terminus of the DMα chain is involved in DR
binding.
Figure 3.12: Measuring DR binding to N-terminally truncated DM using surface plasmon
resonance. In consecutive flow cells 500 RU of DM mutant (αR98A, αR194A), DM with N-terminally
truncated α chain and DM wild type, respectively, were immobilized and DR1/CLIP
low
(2 µM, V8
cleaved) injected for four minutes at a flow rate of 15 μL/min. After buffer injection full length HA
peptide (50 µM) was injected. The experiments were carried out in 50 mM citrate phosphate buffer (pH
5.3), 150 mM NaCl at 25 ºC. The green line displays DR binding to truncated DM (third flow cell) and
the red line DR binding to DM wild type (fourth flow cell). DR binding in the second flow cell (DM
mutant) was subtracted from measurements in the third (DM with N-terminally truncated α chain) or
fourth flow cell (DM wt), respectively. The DR solution consecutively passed through flow chambers one
to four, which can be also the reason why less DR binding was observed for flow cell four.