Cell-permeable small molecule probes for site-specific labeling of proteins.

Article abstract of DOI:10.1039/b309196a

We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells. Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo.

Full text of DOI:10.1039/b309196a

Cell-permeable small molecule probes for site-specific labeling of
proteins†
Dawn S. Y. Yeo,a Rajavel Srinivasan,b Mahesh Uttamchandani,a Grace Y. J. Chen,ab Qing Zhub and
Shao Q. Yao*ab
a Department of Biological Sciences, National University of Singapore, 3 Science Drive 3, Singapore
117543, Republic of Singapore. E-mail: chmyaosq@nus.edu.sg; Fax: 65 6779 1691; Tel: 65 6874 1683
b Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543,
Republic of Singapore. E-mail: chmyaosq@nus.edu.sg; Fax: 65 6779 1691; Tel: 65 6874 1683
Received (in Cambridge, UK) 5th August 2003, Accepted 14th October 2003
First published as an Advance Article on the web 29th October 2003
We have successfully synthesized a number of small
molecule probes designed for site-specific labeling of N-
terminal cysteine-containing proteins expressed in live cells.
Their utility for site-specific, covalent modifications of
proteins was successfully demonstrated with purified pro-
teins in vitro, and with live bacterial cells in vivo.
endogenous molecules, such as cysteine and cystamine, are
present in the cell and will also react with the probe. However,
their reaction products are also small molecules in nature, and
could be easily removed, together with any excessive unreacted
probe, by extensive washing of the cells after labeling. Herein,
we report the detailed synthesis of different cell-permeable
probes, the in vitro labeling studies of selected probes, as well
as their utility in the site-specific labeling of proteins expressed
inside live bacterial cells.
Studying the dynamic movement and interactions of proteins
inside living cells is critical for a better understanding of cellular
mechanisms and functions. Traditionally this has been done by
in vitro labeling of proteins with fluorescent and other
molecular probes, followed by transferring them into a live cell
and monitoring them, in real time and using advanced imaging
techniques such as confocal microscopy, and others.1 Recent
advances in genetic engineering have made it possible to
directly generate fluorescently labeled proteins in living cells,
or even live animals, by fusion of fluorescent proteins such as
GFP (green fluorescent protein) to the protein of interest.2 This
strategy, although extremely powerful, has several problems.
Firstly, the introduction of GFP and other fluorescent proteins to
a target protein may affect its biological and cellular activities,
due to the relatively large size of the fusion (i.e. 27 KDa for
GFP). Secondly, there are currently few fluorescent proteins
available, thereby limiting the number of colors that can be used
to “tag” a protein in vivo. Lastly, the strategy is limited to
protein labeling with fluorophores but not other molecular
probes. In order to address some of these problems, Tsien et al.
recently described a novel method which allows efficient
labeling of proteins in vivo using cell-permeable organoarsenic
compounds.3 Johnsson et al. described enzyme-catalyzed, in
vivo labeling of proteins fused to the human DNA repair protein
hAGT.4 We are interested in developing a novel strategy for
site-specific covalent labeling of proteins, in vivo, by taking
advantage of the chemoselective reaction between thioester-
containing small molecules and proteins expressing N-terminal
cysteines under physiological environments. Previously, N-
terminal cysteine proteins had mostly been used in the semi-
synthesis of proteins, as well as site-specific labeling of proteins
in vitro.5 More recently, the Muir group has successfully
demonstrated, in living cells, the protein semi-synthesis be-
tween two protein fragments fused to the trans-splicing domains
of the Ssp DnaE intein.6
A total of 7 different probes have been synthesized
(Supplementary Information), of which probes 1 to 4 are
fluorophore-containing thioesters (Scheme 1). 5 and 6 are
biotin- and benzophenone-containing probes, respectively. 7 is
a “caged” molecule of 2, in which the fluorescence is designed
to be “turned on” selectively upon photolysis. All probes were
designed to be cell-permeable, in that acetates of different
fluorophores were incorporated in 1, 2 and 4 to increase their
cell permeability. The fluorophore in 3, tetramethylrhodamine
(TMR), as well as the biotin and benzophenone moieties in 5
and 6, respectively, were previously shown to be cell-
permeable.8 Addition of the hydrophobic, benzyl-based thio-
ester in all probes should further increase their cell permeability.
Probes 1 to 4, containing different fluorophores (e.g. coumarin
(CM), fluorescein (FL), TMR and carboxynaphthofluorescein
(CF), respectively) that emit in different colors (e.g. blue, green,
orange/yellow and red, respectively; see Fig. 1), were designed
for potential multicolor cell labeling and imaging. Proteins
labeled with probes 5 and 6 may be used to study protein–
protein interactions by in vivo experiments utilizing biotin–
avidin binding and protein crosslinking, respectively. Probe 7
may be used for protein labeling in a live cell where temporal
and/or confined fluorescence activation is needed.9
In vitro labeling of proteins expressing an N-terminal
cysteine was then carried out with the probes. A model protein,
EGFP (enhanced green fluorescent protein), engineered to
contain an N-terminal cysteine, was incubated with probes 2, 3,
4 and 5 individually, and the extent of protein labeling was
monitored over 24 hours by SDS-PAGE and Western blotting.
8 mM of each probe, with or without 1 mM of DTT, was added
to the pure protein in 1 X PBS buffer. The reaction was
quenched at specified time intervals with 10 mM of cysteine.
Following protein separation on a 12% SDS-PAGE gel, the
labeled protein was visualized and quantitated, either by a
fluorescence gel scanner (in the cases of probes 2, 3 and 4) or
Western blotting using anti-biotin HRP conjugate and Amer-
sham’s ECL kit (in the case of probe 5). Results are summarized
in Fig. 2a and 2b and in the Supplementary Information. In all
cases, the labeling was shown to reach near completion ( > 75%
labeling) within the first 3 hours of the reaction. In addition,
more than 50% labeling occurred within the first 30 min of the
reaction, making this strategy suitable for potential real-time
bioimaging experiments in live cells. The site-specific nature of
the labeling reaction was confirmed by repeating the experiment
under identical conditions with control proteins which either do
not have cysteine residues at all, or have only internal cysteines.
In our strategy (Supplementary Information), a protein of
interest having an N-terminal cysteine is expressed inside a live
cell, by either intein-mediated protein splicing, or ubiquitin
fusion.7 Incubation of the cell with a thioester-containing, cell-
permeable molecule probe allows the probe to efficiently
penetrate through the cell membrane into the cell, where the
chemoselective reaction occurs predominantly between the
thioester and the N-terminal cysteine of the protein. This is true
since endogenous N-terminal cysteine proteins are rare. Other
† Electronic supplementary information (ESI) available: experimental
details and characterization of compounds. See http://www.rsc.org/supp-
data/cc/b3/b309196a/
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Scheme 1 Small molecule probes synthesized and used in the studies.
Fig. 1 Fluorescence spectra of probes 1 (blue), 2 (green), 3 (orange) and 4
(red). Excitation and emission spectra are shown by dashed and solid lines,
respectively. All spectra were taken with 10 mM of dyes in potassium
carbonate buffer. See Supplementary Information for details.
Fig. 2 Site-specific labeling of N-terminal cysteine proteins with the small
In all cases, labeling occurred ONLY with proteins possessing
an N-terminal cysteine (Supplementary Information), thereby
unambiguously supporting our design principle, in which
exclusive labeling should only occur at the N-terminal cysteine
of the target protein. We next applied the strategy to the labeling
of N-terminal cysteine proteins expressed inside live bacterial
cells. E. coli cells overexpressing an N-terminal cysteine GST
(glutathione-S-transferase) protein were treated with probe 3,
and the site-specific labeling of the probe inside the cells was
assessed by fluorescence microscopy and SDS-PAGE (Fig. 2c
and 2d, respectively): exclusive labeling occurred only with E.
coli cells overexpressing N-terminal cysteine GST. Minimal
background labeling was unambiguously confirmed by SDS-
PAGE protein analysis of the labeled cells, indicating that the
labeling occurred predominantly with the N-terminal cysteine
GST.
In conclusion, we have successfully synthesized a number of
cell-permeable, small molecule probes capable of site-specific
labeling of N-terminal cysteine-containing proteins expressed
in live bacterial cells. The strategy should make feasible future
bioimaging experiments where other types of live cells are
involved.
Funding support was provided by the National University of
Singapore (NUS) and the Agency for Science, Technology and
Research (A*STAR) of Singapore.
molecule probes. (a) SDS-PAGE of EGFP labeling with probe 4 for 1 min,
10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h (left to right). For other probes, see
Supplementary Information. (b) % completion of EGFP labeling over
24-hour intervals with probes 2 (blue diamonds), 3 (purple squares), 4
(yellow triangles) and 5 (red diamonds). (c) Fluorescence microscope
images of E. coli expressing N-terminal cysteine GST after labeling with
probe 3 for 24 h. The fluorescence image was overlapped with the phase
contrast image for easy visualization. (d) 12% SDS-PAGE protein analysis
of cells from (c).
Notes and references
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T. J. Tolbert and C. H. Wong, Angew. Chem., Int. Ed., 2002, 41, 2171; (c)
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