27
Chapter I
and the complex was further purified by -DNP affinity chromatography. For
19
F-NMR
experiments DR2/MBP and cleaved DR2/CLIP were both concentrated to a final
concentration of 1-2 mg/mL and buffer exchanged against 50 mM citrate buffer (pH
5.2).
2.2.4
NMR experiments
All NMR experiments with the isotope labeled MBP peptide were conducted on
Bruker spectrometers equipped with cryogenic probes and were transverse relaxation
optimized (tr) (Pervushin et al., 1997). The transverse relaxation-optimized
1
H-
15
N-
labeled heteronuclear single-quantum coherence (tr-HSQC) spectrum of
15
N-labeled
MBP peptide was collected with 0.33 mM of labeled peptide in 20 mM citrate buffer
(pH 5.2) and 10% D
2
O at 30 ºC with a
1
H frequency of 750 MHz and four scans were
measured. The tr-HSQC spectra of isotope labeled MBP peptide in complex with DR2
were measured with a protein complex concentration of 0.35 mM or 0.6 mM in either
10 mM Tris (pH 5.2) and 100 mM NaCl or 50 mM citrate buffer (pH 5.2), both
including 5% D
2
O. The small molecule J10-1 was added at a concentration of 1 mM.
The spectra were collected at various
1
H frequencies (600 MHz, 750 MHz, 900 MHz) at
33 ºC or 37 ºC using Shigemi NMR microtube assemblies (Sigma-Aldrich). For the
three-dimensional,
15
N-selected NOESY-TROSY-HSQC experiment (
15
N-NOESY) a
mixing time of 200 ms was used in order to detect long-range nOes (nuclear Overhauser
effect).
15
N-NOESY and tr-HNCA experiments were measured with a
1
H frequency of
750 MHz. The spectra were processed and analyzed with the programs NMRPipe
(Delaglio et al., 1995), CARA (Keller, 2004) and XEASY (Bartels et al., 1995).
The one-dimensional
19
F-NMR experiments of the small molecule J10-11 were
conducted on a Varian spectrometer with a magnetic field strength of 11.7 Tesla at
25 ºC. The concentration of the small molecule J10-11 was 250 μM in 50 mM citrate
buffer (pH 5.2) and the protein complexes DR2/MBP and DR2/CLIP were added at a
concentration of 11 μM. Micro NMR sample tubes (New Era Enterprises) were used
into which a microcapillary (New Era Enterprises) was inserted. The sample was
pipetted into the microcapillary and 1 mM TFA which was used as a standard with 5%
D
2
O was added between the capillary and the NMR tube. Using microcapillaries
(volume 50 μL) less sample had to be used. The
19
F-NMR experiments were processed
and analyzed using the program MestReNova (Mestrelab Research).