Selective fluorescent chemosensor for the bacterial alarmone (p)ppGpp

Article abstract of DOI:10.1021/ja0759139

We have developed the first selective fluorescent chemosensor (PyDPA) for (p)ppGpp, a bacterial and plant alarmone. By using pyrene-excimer fluorescence, PyDPA shows very good selectivity for (p)ppGpp from among other nucleotides in water. PyDPA was used

Full text of DOI:10.1021/ja0759139

Published on Web 01/01/2008
Selective Fluorescent Chemosensor for the Bacterial Alarmone (p)ppGpp
Hyun-Woo Rhee,† Chang-Ro Lee,‡ Seung-Hyon Cho,‡ Mi-Ryung Song,§ Michael Cashel,|
Hyon E. Choy,§ Young-Jae Seok,‡ and Jong-In Hong*,†
Department of Chemistry, College of Natural Sciences, Seoul National UniVersity, Seoul 151-747, Korea,
Department of Biological Sciences, Seoul National UniVersity, Seoul 151-742, Korea, Department of Microbiology,
Chonnam National UniVersity Medical School, Kwangju 501-746, Korea, and Laboratory of Molecular Genetics,
NICHD, National Institutes of Health, Bethesda, Maryland 20892-2785
Received August 7, 2007; E-mail: jihong@snu.ac.kr
Guanosine 3′-diphosphate-5′-di(tri)phosphate, (p)ppGpp, is a
bacterial and plant alarmone that generates a stringent response
under conditions of nutritional deprivation.1 When bacteria are
placed in stress circumstances such as amino acid starvation, ppGpp
is swiftly synthesized from ATP and GDP by RelA on the bacterial
ribosomal complex. (p)ppGpp is synthesized by the transfer of a
pyrophosphate from ATP to the 3′-OH of GDP (or GTP) (Figure
1). (p)ppGpp influences many bacterial survival mechanisms,1d and
this facilitates bacteria persistence under stress conditions. Further,
(p)ppGpp acts as a global regulator of the gene expression in micro-
Figure 1. Chemical structure of (p)ppGpp.
organisms.2 Therefore, precise and real-time detection of (p)ppGpp
would enable deeper understanding of bacterial physiology.
Since Cashel and Gallant discovered (p)ppGpp in Escherichia
coli,1a (p)ppGpp is usually detected by the isotope (32P) in thin layer
chromatography or by UV/vis absorption of (p)ppGpp in HPLC.3
However, these methods lack sensitivity and are laborious, and
hence, they cannot be applied for the real-time detection of
(p)ppGpp.
Scheme 1. Binding Conformation between PyDPA and ppGpp
Therefore, there is an urgent need to develop selective fluorescent
chemosensors for (p)ppGpp because such chemosensors would
enable the sensitive and real-time detection of (p)ppGpp. To the
best of our knowledge, there has been no report on the development
of fluorescent chemosensors for (p)ppGpp. Here, we report the first
fluorescent chemosensor, PyDPA, which can detect (p)ppGpp
extremely selectively from among other nucleotides in water.
As indicated in Scheme 1, PyDPA consists of two components:
pyrene and bis(Zn2+-dipicolylamine) (dipicolylamine ) DPA). Bis-
(Zn2+-DPA) is well-known for its strong binding to pyrophosphate
groups in water.4 Pyrene was used for its unique excimer emission
(Em ) 470 nm).5
Since the Zn2+-DPA group of PyDPA can bind each pyrophos-
phate moiety at the 3′- and 5′-hydroxyl positions of (p)ppGpp, two
PyDPAs come close to each other upon binding to (p)ppGpp to
generate pyrene-excimer fluorescence due to the stacking of their
pyrene groups (Scheme 1). In fact, our observations were as
expected. Upon the addition of ppGpp (or pppGpp) to PyDPA (20
µM) in water (pH 7.4, 1 mM HEPES buffer, 20 °C), the pyrene-
excimer fluorescence of PyDPA was swiftly enhanced. Job plot
between PyDPA and (p)ppGpp shows 2:1 stoichiometry in water.
When ppGpp (or pppGpp, each 7.0 µM) was added to PyDPA (20
µM) in water, the fluorescence intensity at 470 nm increased
approximately 17- and 14-fold for ppGpp and pppGpp, respectively
(Figure 2A and B).6
(20 µM) in water, there were no pyrene-excimer fluorescence
emissions at 470 nm, only the pyrene monomer fluorescence at
380 nm was enhanced due to the increased hydrophobicity of the
bound NTP or PPi.4d The fluorescence emission ratios (I470nm/I380nm
)
of PyDPA upon the addition of (p)ppGpp, other nucleotides, and
PPi are listed in Figure 2D. Under a UV lamp (excitation at 365
nm), PyDPA emitted strong pyrene-excimer fluorescence only with
(p)ppGpp addition (Figure 2E). These results indicate that PyDPA
can be an extremely selective and sensitive chemosensor that targets
(p)ppGpp from among other nucleotides.
We achieved the real-time fluorescent detection of in vitro ppGpp
synthesis by using extracted E. coli ribosomal complexes (Figure
3). By using ATP (4 mM) and GDP (2 mM), ppGpp synthesis by
the ribosomal complex was detected in real time by the increasing
pyrene-excimer fluorescence of PyDPA (see the Supporting
When other nucleotides (ATP, GTP, CTP, TTP, UTP, cAMP,
or cGMP) and inorganic pyrophosphate (PPi) were added to PyDPA
† Department of Chemistry, Seoul National University.
‡ Department of Biological Sciences, Seoul National University.
§ Chonnam National University Medical School.
| National Institutes of Health.
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J. AM. CHEM. SOC. 2008, 130, 784-785
10.1021/ja0759139 CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. (A) Fluorescence emission titration of PyDPA (20 µM) upon ppGpp addition; Ex ) 344 nm, Em ) 470 nm. (B) Fluorescence emission titration
of PyDPA (20 µM) upon pppGpp addition; Ex ) 344 nm, Em ) 470 nm. (C) Fluorescence emission spectra of PyDPA upon the addition of 7 µM of each
nucleotide and PPi; Ex ) 344 nm. (D) Fluorescence emission intensity ratio (I470nm/I380nm) of PyDPA (20 µM) upon the addition of ppGpp, pppGpp, and
other nucleotides (each 7 µM). ppGpp and pppGpp are depicted on the graph, and the others are numbered in panel C. (E) Fluorescence emission of PyDPA
(20 µM) under a UV lamp (excitation wavelength ) 365 nm) upon the addition of ppGpp, pppGpp, ATP, and GTP (left to right, each 7 µM).
Supporting Information Available: Synthesis of PyDPA, Job’s
plot between PyDPA and (p)ppGpp, PPi competition experiment results
with (p)ppGpp, fluorescence emission titration of PyDPA with more
than 7.7 µM (p)ppGpp, experimental data for the detection of in vitro
ppGpp synthesis by extracted bacterial ribosomal complexes, experi-
mental data for the detection of internal ppGpp in the starved bacterial
cells. This material is available free of charge via the Internet at http://
pubs.acs.org.
References
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PyDPA can also be used to detect ppGpp produced in the starved
bacterial cells.9 As shown in Figure S6, bacterial ppGpp can be
detected either by HPLC (UV absorption) or by PyDPA. However,
the fluorescent detection by PyDPA was much more selective and
sensitive than the HPLC method.
In summary, we have developed the first fluorescent chemosensor
(PyDPA) for the detection of (p)ppGpp, a bacterial and plant
alarmone. PyDPA can detect (p)ppGpp extremely selectively from
among other nucleotides or pyrophosphate in water by using pyrene-
excimer fluorescence. PyDPA was used for the real-time detection
of in vitro ppGpp synthesis by the E. coli ribosomal complex.
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Acknowledgment. This work was supported by the KRF (Grant
No. KRF-2006-312-C00592) and Seoul R&BD. This research was
also supported in part by the NICHD Intramural Research Program
at NIH and Chung grant. H.-W.R. is the recipient of the Seoul
Science Fellowship.
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