Supporting Information
Wiley-VCH 2010
69451 Weinheim, Germany
Detection and Identification of Protein-Phosphorylation Sites in
Histidines through HNP Correlation Patterns**
Sebastian Himmel, Sebastian Wolff, Stefan Becker, Donghan Lee,* and Christian Griesinger*
anie_201003965_sm_miscellaneous_information.pdf
Supporting material
Histidine phosphorylation
Mixtures of 4.1 mg 15N3 L-histidine (Isotec, 95% 15N) and 2.2 mg phosphoramidate (PA) synthesized as described
previously[1] were dissolved in 250 µl of 40 mM sodium phosphate buffer containing 10% D2O, 5 mM MgCl2 and 2 mM
dithiothreitol (DTT). The reaction took place for one hour at room temperature before it was filled into a 5 mm NMR sample
tube.
Purification and phosphorylation of proteins
The Bacillus subtilis phosphocarrier protein (HPr) coding DNA was cloned into pET16bTEV (modified Novagene pET16b
with TEV cleavage site) expression vector and expressed at 37°C for 12 h in Escherichia coli BL21 (DE3) (Stratagene). The
Bacillus subtilis PRDI coding sequence was cloned into pGEX-2TEV expression vector (modified GE Healthcare pGEX-2T,
with TEV cleavage site) and expressed at 22°C overnight in Escherichia coli BL21 (Stratagene). The uniformly 15N-labeled
proteins were grown in M9 minimal medium supplemented with 15NH4Cl. The His-tagged HPr was purified on Ni-NTA
agarose (Qiagen) whereas the GST-tagged PRDI was purified on Glutathione Sepharose 4B (GE Healthcare). Both proteins
were digested by Tobacco Etch Virus (TEV) protease, followed by gel filtration (GE Healthcare SD 75, 16/60) in 200 mM
NaCl, 50 mM Tris HCl, 5 mM MgCl2, 2 mM dithiothreitol (DTT) and pH 7.4. Pooled elution fractions were dialysed against
NMR buffers 40 mM sodium phosphate (pH 7.4 for HPr; pH 7.3 for PRDI) containing 5 mM MgCl2 and 2 mM DTT.
Phosphorylation of HPr was achieved by adding Enzyme I (EI) in a molar ratio of 1/30 compared to HPr and
phosphoenolpyruvate (trisodium salt hydrate, Sigma-Aldrich) in 76 molar excess respectively. In vitro phosphorylation of
PRDI will be published elsewhere. Unlabeled His-tagged EI was overexpressed in BL21 (DE3) at 37 °C and after sonication
purified on Ni-NTA agarose. No protease digestion was applied before gel filtration (SD 75, 16/60) in 200 mM ammonium
acetate, 2 mM DTT and pH 7.4. Final protein concentrations of HPr and PRDI were ~ 1 mM.
pH titration of phosphohistidine
pH titration was carried out with two samples. 3.2 and 3.7 mg 15N3 L-histidine were mixed with 2.9 and 3.0 mg of PA in
200 µl und 182.5 µl (180 µl H2O and 2.5 µl 5 M NaOH) of H2O respectively. The phosphorylation reaction took place at room
temperature. Both samples were titrated by either adding small amounts of 1 M and 5 M NaOH or 18.5% HCl respectively.
The pH was monitored before and after every NMR measurement step.
NMR spectroscopy
All NMR experiments were performed at 303 K with a 1H resonance frequency of 600 MHz. pH titrations measured by 1D
31
P experiments were recorded on a Bruker Avance III spectrometer equipped with a QXI probe head. Spectra of HNP
experiments for phosphohistidines, HPr and PRDI were recorded on a Bruker Avance II spectrometer equipped with a QCI
probe head, accumulating 4, 24 and 216 scans, respectively. Spectra were processed and analyzed with the nmrPipe package[2]
and TOPSPIN 2.0 (Bruker Biospin). 3D HNP spectrum of HPr was recorded with 15 (31P), 35 (15N), and 512 (1H) complex
points with t1max= 7.7 ms, t2max= 8.2 ms, and t3max = 65.8 ms within ~ 20 h. Data of PRDI were collected within ~ 67 h and 13
(31P), 15 (15N), and 512 (1H) complex points with t1max= 6.7 ms, t2max= 8.2 ms, and t3max = 65.8 ms, respectively.
Figure S1. A contour plot of the 1H,15N projection from the 3D HNP
spectrum of phosphocarrier protein HPr from Bacillus subtilis at pH 7.4
and 303 K. A “one peak pattern” points to a phosphorylated δ1 site
corresponding to 1.
References
[1]
[2]
D. R. Buckler, A. M. Stock Anal. Biochem. 2000, 283, 222-227.
F. Delaglio, S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, A. Bax J. Biomol. NMR 1995, 6, 277-293.