Biophysical properties of hGBP5a ⁄ b and hGBP5ta
M. Wehner and C. Herrmann
Elution was followed by monitoring the absorbance at
¨
80 nm using an Akta Purifier system (GE Healthcare).
References
2
1
2
3
Bourne HR, Sanders DA & McCormick F (1990) The
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Fluorescence titration
Bourne HR, Sanders DA & McCormick F (1991) The
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Fluorescence titrations were performed at 25 ꢀC using an
SFM25 fluorospectrometer (Kontron, Zurich, Switzerland)
and mant-labeled nucleotides (Jena Biosciences). The excita-
tion and emission wavelengths were 366 and 435 nm,
respectively. mant-labeled nucleotide (0.5 lm) was titrated
using protein solutions (typically approximately 100 lm)
containing 0.5 lm of the mant-labeled nucleotide to avoid
dilution of the fluorophore. The data were analysed using a
quadratic binding equation as described previously [19].
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binding protein (GBP) gene clusters. J Interferon
Cytokine Res 26, 328–352.
Stopped flow measurements
All measurements were performed using an SFM400
stopped flow apparatus (Bio-Logic, Claix, France). For
association kinetic experiments, 38 lL of a nucleotide solu-
tion (0.5 lm) and 38 lL of hGBP5a ⁄ b or hGBP5ta
6
7
Cheng YS, Colonno RJ & Yin FH (1983) Interferon
induction of fibroblast proteins with guanylate binding
activity. J Biol Chem 258, 7746–7750.
(
increasing concentrations starting from a 10-fold molar
Schwemmle M & Staeheli P (1994) The interferon-
induced 67-kDa guanylate-binding protein (hGBP1) is a
GTPase that converts GTP to GMP. J Biol Chem 269,
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excess) were mixed at 14 mLÆs . In the case of mant-GTP
association, it was necessary to use increasing concentra-
tions of the nucleotide because of nucleotide hydrolysis
1
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(0.5 lm of protein was mixed with increasing concentra-
8
9
Praefcke GJ, Geyer M, Schwemmle M, Kalbitzer HR &
Herrmann C (1999) Nucleotide-binding characteristics
of human guanylate-binding protein 1 (hGBP1) and
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tions of mant-GTP starting from a 15-fold molar excess).
Fluorescence was excited at 295 nm (mant-GTP) or
3
66 nm (all others), and recorded using a 420 nm cut-off
filter. For mant-GTP, we used a fluorescence resonance
energy transfer approach (primary excitation of the trypto-
phans in the G-domain and transfer to the mant-group of
the nucleotide) to minimize excitation of unbound nucleo-
tide and thereby loss of signal quality. The traces were
fitted using a single rate constant, and the resulting rates
Kunzelmann S, Praefcke GJ & Herrmann C (2006)
Transient kinetic investigation of GTP hydrolysis
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8627–28635.
(kobs) were plotted against the protein or mant-GTP con-
1
1
0 Anderson SL, Carton JM, Lou J, Xing L & Rubin BY
centration. Using a linear fit, the association rate constants
were extracted from the slope (kon), and the intercept rep-
resents the dissociation rate (koff). In the case of displace-
ment experiments, 10 lm protein was preincubated with
(
(
1999) Interferon-induced guanylate binding protein-1
GBP-1) mediates an antiviral effect against vesicular
stomatitis virus and encephalomyocarditis virus.
Virology 256, 8–14.
0
.5 lm of mant-nucleotide. The mant-nucleotide was dis-
placed by mixing with a 1000-fold excess of unlabeled
GDP, and the resulting rate constant corresponds to kdiss
Fluorescence traces were fitted using a single rate constant
mant-GDP) or two rate constants (mant-GTPcS). Corre-
sponding dissociation constants are calculated from the
relationship K
1 Itsui Y, Sakamoto N, Kurosaki M, Kanazawa N,
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(
6
90–700.
d
= koff ⁄ kon.
1
2 Tietzel I, El-Haibi C & Carabeo RA (2009)
Human guanylate binding proteins potentiate the
anti-chlamydia effects of interferon-c. PLoS ONE 4,
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Acknowledgements
¨
We thank Professor Dr Michael Sturzl for providing
1
3 Guenzi E, To
C, Jorg A, Matzen K, Zietz C, Kremmer E, Nappi F,
Schwemmle M et al. (2001) The helical domain of
¨
polt K, Cornali E, Lubeseder-Martellato
the cDNA for hGBP5, the Deutsche Forschungsgeme-
inschaft for financial support, and the Ruhr-University
Research School for a full scholarship to M.W.
¨
1
604
FEBS Journal 277 (2010) 1597–1605 ª 2010 The Authors Journal compilation ª 2010 FEBS