XIII
Table
of contents
2.3.1
Preparing and crystallizing HLA-DR2/MBP in the presence of J10-1 or J10-12 ......... 28
2.3.2
Preparing and characterizing
15
N-labeled MBP peptide ................................................ 29
2.3.3
Preparing the complex of HLA-DR2 and
15
N-labeled MBP peptide ............................ 31
2.3.4
Measuring
1
H-
15
N HSQC spectra of
15
N-labeled MBP peptide free in solution and in
complex with HLA-DR2 ............................................................................................... 31
2.3.5
Deuterating MBP peptide to improve signal intensity of the HSQC spectrum of isotope
labeled MBP peptide in complex with HLA-DR2 ........................................................ 35
2.3.6
Measuring and comparing HSQC spectra of HLA-DR2/
15
N,
13
C,
2
H-MBP before and
after addition of J10-1 ................................................................................................... 36
2.3.7
Performing HNCA and
15
N-NOESY experiments of HLA-DR2/
15
N,
13
C,
2
H-MBP to
determine resonance assignments ................................................................................. 39
2.3.8
Measuring
19
F-NMR spectra of J10-11 in the presence of HLA-DR2/MBP and HLA-
DR2/CLIP ..................................................................................................................... 40
2.4
Conclusion ............................................................................................................ 42
3 Chapter II: Structural effects of destabilizing peptide-MHC II interactions and
implications for the peptide exchange mechanism of HLA-DM ......................... 46
3.1
Introduction ......................................................................................................... 46
3.2
Materials and Methods ....................................................................................... 47
3.2.1
Preparation of biotinylated HLA-DM and HLA-DM mutant ........................................ 47
3.2.2
Preparation of HLA-DM used for crystallization .......................................................... 48
3.2.3
Preparation of covalently linked HLA-DR1/peptide complexes and of HLA-DR1
mutant ............................................................................................................................ 48
3.2.4
Preparation of HLA-DM with an N-terminally truncated α chain ................................ 49
3.2.5
Surface plasmon resonance experiments ....................................................................... 49
3.2.6
Protein crystallization, collection of diffraction data and structure solving .................. 50
3.2.7
Eluting covalently linked peptide from HLA-DR1 ....................................................... 51
3.2.8
Mass spectrometry ........................................................................................................ 51
3.3
Results and discussion ......................................................................................... 51
3.3.1
Preparing HLA-DM and HLA-DR1 carrying a truncated HA peptide, both used for
crystallization experiments ............................................................................................ 51
3.3.2
Measuring pH-dependent affinity of HLA-DM to HLA-DR1 carrying an N-terminally
truncated HA peptide using surface plasmon resonance ............................................... 53
3.3.3
Crystallizing HLA-DR1 carrying a truncated HA peptide, in the presence of HLA-DM
and alone ....................................................................................................................... 55
3.3.4
Solving the structure of HLA-DR1 carrying an N-terminally truncated HA peptide .... 56
3.3.5
Structure of HLA-DR1 carrying an N-terminally truncated HA peptide ...................... 57
3.3.6
Eluting N-terminally truncated HA peptide from HLA-DR1 protein used for
crystallization and analyzing by mass spectrometry ..................................................... 65