A biocompatible condensation reaction for the labeling of terminal cysteine residues on proteins

Article abstract of DOI:10.1002/anie.200903627

Going live: A protein-labeling method based on the use of a single amino acid taglan N-terminal cysteine residuel and small-molecule probes containing a cyanobenzothiazole (CBT) unit has been used for the specific fluorescence labeling of proteins in vitro and at the surface of live cells (see scheme). This simople ligation reaction proceeds with a high degree of specificity under physiological conditions. Rd: a rhodamine dye; TEV: Tobacco etch virus.

Full text of DOI:10.1002/anie.200903627

Communications
DOI: 10.1002/anie.200903627
Protein Labeling
A Biocompatible Condensation Reaction for the Labeling of Terminal
Cysteine Residues on Proteins**
Hongjun Ren, Fei Xiao, Ke Zhan, Young-Pil Kim, Hexin Xie, Zuyong Xia, and Jianghong Rao*
The site-specific labeling of
proteins with molecular
tags enables the direct vis-
ualization
of
protein
dynamics, localization, and
interactions in single living
cells and is a powerful tool
for studying the structure
[1]
and function of proteins.
Proteins of interest can be
labeled by genetic fusion to
fluorescent proteins or
chemical reactions with flu-
orescent dyes. For chemical
labeling, a receptor protein
is often used, for example, a
6
mutant of human O -alkyl-
guanine-DNA transfera-
[
2a]
se
or Escherichia coli
[2b]
dihydrofolate reductase,
which binds to or reacts
with a fluorescently tagged
[2]
ligand.
Alternatively,
smaller tags, such as short
peptides, can be labeled by
selective binding to fluoro-
Scheme 1. Condensation reactions between cysteine/N-terminal cysteine residues and CBT derivatives in the
synthesis of d-luciferin and site-specific protein labeling, and structures of the CBT probes used for labeling.
PBS=phosphate-buffered saline.
[
3]
genic dyes or by enzyme-
catalyzed ligation to fluo-
[
4]
rescent probes. Water-compatible chemical reactions can
also be applied to protein labeling, such as the Staudinger
N-Terminal cysteine residues have frequently been used
in protein engineering for site-specific labeling and modifi-
cation. Thioesters are commonly used in a ligation reaction
[
5]
[8]
reaction between azides and triphenylphosphane, the Huis-
gen cycloaddition or “click reaction” between azides and
with terminal cysteine residues that proceeds through trans-
[
6]
[9]
alkynes, or reactions between aldehydes (or ketones) and
thioesterification and S-to-N acyl exchange. This native
[7]
aminooxy-containing reagents (or hydrazides). Herein, we
describe a water-compatible condensation reaction for the
labeling of terminal cysteine residues on proteins in vitro and
at the cell surface.
chemical ligation reaction has been applied successfully to
[10]
protein semisynthesis and labeling.
Our method to label the terminal cysteine residue of a
protein is based on the condensation of 2-cyanobenzothiazole
(CBT) and d-cysteine. This reaction, which is used as the last
step of the synthesis of d-luciferin, a common substrate for
[
11]
[*] Dr. H. Ren, F. Xiao, K. Zhan, Y.-P. Kim, H. Xie, Z. Xia, Prof. J. Rao
firefly luciferase (reaction 1 in Scheme 1),
can proceed
Molecular Imaging Program at Stanford
Departments of Radiology and Chemistry, Stanford University
smoothly in aqueous solutions. We hypothesized that CBT
could react with the terminal cysteine residue of a protein. If a
fluorophore was conjugated to the CBT motif, this reaction
should enable the ligation of a fluorescent label specifically to
the terminal cysteine residue of the protein (reaction 2 in
Scheme 1).
1201 Welch Road, Stanford, California 94305-5484 (USA)
Fax: (+1)650-736-7925
E-mail: jrao@stanford.edu
Homepage: http://raolab.stanford.edu
[
**] This research was supported by a grant from NIGMS
We first investigated whether CBT could react with
functional groups other than free cysteine. With homocys-
teine, which contains a 1,3-aminothiol group, a stable
condensation product with a six-membered ring was gener-
(
R01GM086196-01). We thank Prof. Matthew Bogyo at Stanford for
access to the mass spectrometry facility.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200903627.
9
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ated. As expected, when the thiol group was replaced with a
hydroxy group, for example, in b-amino alcohols and serine,
no detectable products were formed under similar conditions.
When glutathione or b-mercaptoethanol was mixed with
2
-cyano-6-aminobenzothiazole (amino-CBT) at a molar ratio
of 2:1, a new peak was detected by HPLC analysis after
0 minutes besides that of the remaining amino-CBT (50%).
3
However, both of these peaks disappeared after the further
addition of free l-cysteine, and only the condensation product
l-aminoluciferin was observed. This result suggests that CBT
can react reversibly with free thiol groups. The reaction is
selective for 1,2-aminothiol (or 1,3-aminothiol) substrates
over simple thiol groups. Indeed, when amino-CBTwas mixed
with cysteine and glutathione (or 2-mercaptoethanol) at a
molar ratio of 1:5:5, only the condensation product l-amino-
luciferin was observed by HPLC analysis (see Figure S2 in the
Supporting Information).
We evaluated whether aromatic cyano compounds other
than CBT could similarly react and cyclize with free cysteine.
Both benzonitrile and picolinonitrile failed to produce
detectable products under the same conditions. A mixture
of picolinonitrile and amino-CBT (1:1) with free l-cysteine
only afforded l-aminoluciferin, as determined by HPLC
Figure 1. In vitro labeling of N-terminal cysteine residues on proteins
with CBT probes. a) Generation of an N-terminal cysteine residue
through protease processing. b) Fluorescence (left) and white-light
analysis. The second-order rate constant for this reaction was
À1 À1
determined to be 9.19m
s
(see Figure S4 in the Supporting
(right) images of a gel loaded with proteins (10 mm) labeled with
Information), which is significantly larger than the value
amino-CBT (50 mm; lane 1: BSA; lane 2: lysozyme; lane 3: rLuc;
lane 4: Cys-rLuc) or FITC–CBT (lane 5: Cys-rLuc) and stained with
Coomassie Blue (lane 6: size markers). All reactions were quenched
with free cysteine before gel loading.
reported for
a
biocompatible click reaction (7.6 ꢀ
À2 À1 À1 [12]
1
0 m s ).
Before applying the reaction to protein labeling, we tested
the labeling method with cysteine-containing peptides. Sev-
eral peptides with an N-terminal l-cysteine residue were
synthesized. HPLC analysis indicated that they all underwent
conjugation with amino-CBT in a phosphate buffer at pH 7.4
at room temperature in more than 90% yield within
observed on the gel for three control proteins without an N-
terminal cysteine residue—bovine serum albumin (BSA),
lysozyme, and unmodified luciferase—even though all con-
tained cysteine residues in their sequences (Figure 1b,
lanes 1–3). This result demonstrates that the ligation takes
place specifically with N-terminal cysteine residues.
A biotinylated CBT probe was prepared for labeling Cys-
rLuc (Scheme 1). A similar reaction afforded biotinylated
Cys-rLuc with a measured molecular weight of 37330 Da (the
calculated MW is 37336 Da; see Figure S5 in the Supporting
Information). The biotinylated Cys-rLuc was able to bind
streptavidin to form the expected complex, as revealed by gel
electrophoresis (see Figure S6 in the Supporting Informa-
tion).
3
0 minutes; the identity of each product was confirmed by
mass spectrometry (see Table S1 in the Supporting Informa-
tion). For peptides with the cysteine residue in the middle of
the sequence, no ligation product was detected by HPLC
analysis, which suggests that CBT specifically labels N-
terminal cysteine residues of peptides.
We next tested this method to label a cysteine residue at
the N terminus of the bioluminescent protein Renilla lucifer-
ase. Both proteolytic processing and the spontaneous hydrol-
ysis of intein fusion protein have been reported to generate an
[8c,10c]
N-terminal cysteine residue on a protein.
peptide substrate of tobacco etch virus (TEV) protease
ENLYFQflC; arrow indicates the cleavage site) to the
We fused the
A free cysteine residue may be introduced at the
C terminus of rLuc by the intein-mediated cleavage reaction
for CBT labeling (Figure 2a). A recombinant protein con-
taining rLuc and Mex GyrA intein (Mycobacterium xenopi
(
N terminus of Renilla luciferase (Figure 1a). Furthermore, a
six-histidine tag sequence was added in front of the TEV
protease substrate to facilitate purification. The fusion
[13]
gyrase A intein, a 198 aa natural mini intein which lacks a
central intein endonuclease domain) was expressed and
purified. The GyrA intein catalyzed the formation of the
thioester intermediate between the N terminus of GyrA and
the C terminus of rLuc. The addition of thiol nucleophiles,
such as l-cysteine or dicysteine (two carboxylate groups are
linked by an ethyldiamine moiety), resulted in the cleavage of
the thioester to generate a free GyrA species and rLuc with
the nucleophile conjugated at its C terminus. With l-cysteine,
the product rLuc-Cys only contained a free thiol group, but
2
+
protein was expressed and purified on a Ni /nitrilotriacetic
acid (NTA) column. TEV protease was then added to cleave
the substrate, and elution afforded the product N-terminal-
cysteine luciferase (Cys-rLuc).
When Cys-rLuc was incubated with amino-CBTor FITC–
CBT (FITC = fluorescein isothiocyanate) at room temper-
ature for two hours, a fluorescent ligation product of the
expected size was observed clearly on the gel (Figure 1b,
lanes 4 and 5). In comparison, no labeling products were
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Figure 2. In vitro labeling of cysteine residues introduced at the C terminus of proteins with CBT probes. a) Introduction of free cysteine residues
at the C terminus of rLuc through intein-mediated splicing; a linker peptide is present between rLuc and GyrA. b) Fluorescence (right) and white-
light (left) images of a gel loaded with reaction solutions (lane 1: rLuc-Cys; lane 2: rLuc-diCys; lane 3: rLuc-Cys+peptide–FITC–CBT; lane 4: rLuc-
diCys+peptide–FITC–CBT) and stained with Coomassie Blue.
with dicysteine, the product rLuc-diCys also contained a free
cysteine residue at the C terminus available for CBT labeling.
Both l-cysteine and dicysteine cleaved the thioester effi-
ciently and gave the expected products (Figure 2b, lanes 1
and 2). When the two reaction solutions, containing 5 mm
protein, were incubated with a CBT probe (peptide–FITC–
CBT at 50 mm; see Figure S7 in the Supporting Information),
free GyrA formed a fluorescent adduct detectable on the gel
because it contained an N-terminal cysteine residue (Fig-
ure 2b, lanes 3 and 4); on the other hand, fluorescent staining
was observed for rLuc-diCys (lane 4) but not for rLuc-Cys
(
lane 3). No fluorescent staining on the gel was observed for
the uncleaved fusion protein that remained in the reaction
mixture either, which suggested that the thiol group of the
cysteine residue at the reactive site of the fused GyrA protein
did not react with CBT. This result again showed that a
terminal cysteine residue on a protein can be labeled
specifically by the CBT reaction.
The labeling kinetics of GyrA was measured by gel
analysis of the reaction mixture with different incubation
times (Figure 3a). Consistent with the fast kinetics observed
with free cysteine, the labeling of GyrA by amino-CBT was
nearly 50% complete after the first 10 minutes and 75%
complete after 150 minutes (Figure 3b). The labeling of free
cysteine at the C terminus of rLuc-diCys was nearly complete
Figure 3. Time-dependent labeling of the N-terminal cysteine residue
of GyrA. a) Fluorescence (left) and white-light (right) images of a gel
loaded with reaction solutions containing GyrA (7.1 mm) and amino-
CBT (57 mm) at different reaction times (lane 1: 0 min; lane 2: 10 min;
lane 3: 30 min; lane 4: 1 h; lane 5: 2.5 h; lane 6: 12 h) and stained
with Coomassie Blue (lane 7: size markers). The reactions were
quenched with cysteine before gel loading. b) Plot of integrated
fluorescence-band intensity versus time.
(
conversion almost 100%) after one hour when 4 equivalents
of the CBT probe were used (see Figure S8 in the Supporting
Information).
We were interested in whether this condensation reaction
could be applied to the specific labeling of proteins in vivo.
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Cyan fluorescent protein (CFP) was used as a model target
and expressed on the cell surface. TEV protease was used to
generate an N-terminal cysteine residue through the proteo-
lytic processing of the TEV protease substrate fused to the
N terminus of CFP. To ensure that the fusion protein was
targeted on the extracellular side of the cell membrane, the
murine immunoglobulin (Ig) k-chain leader sequence was
added immediately before the N terminus of the substrate
sequence of TEV protease, and its C terminus contained the
platelet derived growth factor receptor (PDGFR) transmem-
brane domain. TEV protease treatment would remove the
murine Ig k-chain leader sequence and the substrate fragment
containing the ENLYFQ sequence, and expose the free N-
terminal cysteine residue to labeling with the rhodamine–
CBT probe (Figure 4a).
We first verified the labeling of the fusion protein in cell
lysates. HeLa cells were transfected with the CFP construct
and harvested for lysis. The collected supernatant was
incubated with the rhodamine–CBT probe in the presence
or absence of TEV protease. Gel analysis revealed a
fluorescent band corresponding to the size of the CFP
fusion protein (34 kDa) in the presence of TEV protease;
however, no band was detected in the absence of TEV
protease (Figure 4b). Furthermore, no other fluorescent
bands were observed on the gel. This result confirmed the
specificity of the labeling.
Live transfected HeLa cells were incubated with the
labeling solution containing both the rhodamine–CBT probe
and TEV protease for 30 minutes at 378C. Subsequent
washing removed the unreacted probe before imaging. Live-
cell fluorescence imaging in the CFP channel confirmed the
membrane expression of the fusion protein (Figure 4c). Clear
membrane staining by the probe was observed in the rhod-
amine channel and was almost perfectly superimposable with
that observed in the CFP channel (Figure 4c). Typically, more
than half of the cells were labeled by the rhodamine–CBT
probe by this procedure. A control staining without TEV
protease in the labeling solution gave little detectable
fluorescence (Figure 4d). Cells transfected with nontagged
CFP were not labeled either. Similar labeling was carried out
successfully with other probes on other cell lines, such as
COS-7 (see Figure S10 in the Supporting Information). These
results demonstrate that the condensation reaction can take
place in the context of live cells with the specific labeling of
the N-terminal cysteine residue of target proteins on the cell
surface.
The condensation reaction is mechanistically different
from the thioester-based ligation, and the CBT probes show
good stability and selectivity for 1,2-aminothiols over thiol
groups without an adjacent amino group. An excess of the
thiol is often added to facilitate the thioester-based ligation
but is unnecessary for the CBT condensation reaction.
Figure 4. Labeling of CFP on live HeLa cells with rhodamine–CBT. a) Schematic illustration of the labeling strategy. b) Analysis by gel
electrophoresis of the in vitro labeling of cell lysates with rhodamine–CBT (lane 1: size markers; lane 2: cell lysates without TEV protease; lane 3:
cell lysates with TEV protease ; lanes 4 and 5: fluorescence image of lanes 2 and 3, respectively). c) Labeling of tagged CFP in transfected HeLa
cells with TEV protease (10 units) and rhodamine–CBT (2 mm). Left: CFP fluorescence; middle: rhodamine fluorescence; right: an overlay of left
and middle images. d) Images of HeLa cells transfected with tagged CFP under the same conditions as in (c) but without TEV protease.
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Furthermore, in vivo labeling proceeds much faster than
reported examples of thioester-based labeling, which required
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2
4 hours as opposed to only 30 minutes in our study.
Although this labeling method is limited to the terminal
cysteine residue of a target protein, the small tag size (with
just one amino acid) should offer an important advantage.
The simple procedure for live-cell labeling and the short
labeling time are also attractive. A free cysteine residue at the
C terminus may be labeled as well, although its generation
requires extra chemical modifications that are less straight-
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fragment with an N-terminal cysteine residue may be ligated
to another fragment containing a CBT moiety that has been
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existing approaches to protein labeling, this simple conden-
sation reaction is compatible with physiological conditions
and proceeds with a high degree of specificity and efficiency.
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Received: July 3, 2009
Revised: September 24, 2009
Published online: November 18, 2009
Keywords: chemical ligation · condensation ·
.
fluorescent probes · live-cell imaging · protein modifications
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