29
Chapter I
Table 2.1: Data collection and refinement statistics of DR2/MBP. The data set was collected at
beamline X25 at the National Synchrotron Light Source (Brookhaven Laboratories, Upton, NY, USA).
The crystal was grown in 100 mM glycine (pH 5.2), 15 % PEG 6,000 and 20 mM sodium acetate at
18 ºC. Values in parenthesis refer to the highest resolution shell.
Data collection
Space group
P3
1
Cell dimensions
a, b, c (Å)
119.2, 119.2, 76.2
Resolution (Å)
50.0-2.90 (2.95-2.90)
R
merge
(%)
8.4 (44.1)
I/σI
35.0 (2.5)
Completeness (%)
98.9 (90.5)
Redundancy
4.9 (2.9)
Refinement
Resolution (Å)
42-2.90
No. unique reflections
26527 (1229)
R
work
/R
free
(%)
22.4/27.5
Number of atoms (ASU)
Protein
6116
Peptide
264
Water
183
B-factors (Å
2
)
Protein
83.5
Peptide
88.2
Water
85.3
R.m.s. deviations
Bond lengths (Å)
0.008
Bonds angles (º)
1.399
2.3.2
Preparing and characterizing
15
N-labeled MBP peptide
As the small molecule might have bound to only a subset of DR molecules in the co-
crystal structure and might have a fast on/off rate, NMR spectroscopy was pursued to
investigate small molecule binding in solution. To track MBP peptide bound to DR2
upon small molecule binding the peptide was labeled with different isotopes. For
isotope labeling, the 15 amino acid long MBP peptide (ENSVVHFFKNIVTSR) was
expressed separately from the DR protein (as a trpLE fusion protein). The peptide
sequence corresponds to the MBP protein sequence 85-99 except that two prolines at
positions three and 14 were substituted with serine. Prolines were replaced as they
generate no
resonances in
1
H-
15
N HSQC experiments due to absence of amide protons.
For the initial NMR experiments the MBP-peptide was
15
N-labeled and the yield of
the peptide together with the fusion protein was 140 mg per 1 liter bacterial culture. The
peptide together with the fusion protein was purified following the preparation scheme
shown in figure 2.2. Using SDS-PAGE it was determined that ~ 70% of the MBP-
fusion protein was cleaved using CNBr. The correct mass of the purified peptide was