Thiol-yne radical reaction mediated site-specific protein labeling via genetic incorporation of an alkynyl-l-lysine analogue

Article abstract of DOI:10.1039/c3ob27116a

Three alkyne-containing pyrrolysine derivatives were synthesized and genetically encoded into proteins by a mutant PylRS-tRNA pair with high efficiencies. With these alkyne handles, site-specific dual labeling of proteins can be achieved via a bioorthogonal thiol-yne ligation reaction.

Full text of DOI:10.1039/c3ob27116a

Organic &
Biomolecular Chemistry
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PAPER
Thiol–yne radical reaction mediated site-specific
protein labeling via genetic incorporation of an
alkynyl-L-lysine analogue†
Cite this: Org. Biomol. Chem., 2013, 11,
624
2
a,b
a,c
a,c
a,c
b
Yiming Li,* Man Pan, Yitong Li, Yichao Huang and Qingxiang Guo
Received 30th October 2012,
Accepted 31st January 2013
Three alkyne-containing pyrrolysine derivatives were synthesized and genetically encoded into proteins
by a mutant PylRS–tRNA pair with high efficiencies. With these alkyne handles, site-specific dual labeling
of proteins can be achieved via a bioorthogonal thiol–yne ligation reaction.
DOI: 10.1039/c3ob27116a
www.rsc.org/obc
7
tolerate a variety of functional groups. In our previous work,
the TEC reaction was used for site-specific dual labeling of
Introduction
Site-specific labeling and modification of proteins have seen proteins carrying two site-specifically incorporated alkene
1
8
tremendous development in the last decade. In addition handles. However, the incorporation efficiency of unnatural
to the well established bioorthogonal reactions such as amino acids (UAA) was relatively low which limited its appli-
Staudinger ligation, azide–alkyne cycloaddition, photo- cation. We thus anticipate that TYC may be more suitable
inducible tetrazole-containing 1,3-dipolar cycloaddition and for peptide/protein dual labeling and modification via the UAA
2
native chemical ligation, the radical mediated thiol–ene and approach.
thiol–yne coupling (TEC and TYC) procedures have recently
Till now, very few TYC-mediated peptide/protein dual label-
3
emerged as a new metal-free bioorthogonal reaction. In ing reactions have been developed although these reactions
principle, TEC and TYC are anti-Markovnikov regioselective are considered a good option. Recently, Dondoni et al. used
reactions induced by photo-irradiation via a radical initiator. TYC to achieve selective propargylation of cysteine-containing
They have been successfully used for polymer–polymer conju- peptides by coupling with glycosyl thiols for post-translational
9
gation, materials surface modification and peptide/protein dual glycosylation of peptides. Moreover, Dondoni and Davies
4
labeling. The fact that the TEC and TYC reactions can be per- groups reported the TYC-mediated multi-glycoconjugation and
formed with low-energy light at 365–400 nm and their robust- fluorescent labeling of serum albumin induced by light at
1
0
ness in aqueous buffer contribute to their promising visible wavelength (365–405 nm). Furthermore, the Boelens
5
applications, especially in biological studies.
group demonstrated that thiol–yne addition with reduced pep-
Among the two radical mediated coupling reactions, photo- tidylcysteines is responsible for most of the azide-independent
1
1
induced hydrothiolation of alkynes (TYC) couples two thiols to polypeptide labeling. Despite these developments, we need
one alkyne under UV (365 nm) irradiation at ambient tempera- to note that the previous TYC-based protein dual labeling
ture. Compared to TEC, TYC allows simple addition of two strategies have a limitation, that is, the target protein should
thiols to an alkyne for dual ligation and has been introduced contain only one free cysteine residue unless one wants to
3
c
for the development of a cross-linked polymer network.
conduct random labeling at multiple locations. To this end,
Moreover, TYC is usually more efficient when carried out with redundant cysteine residues must be mutated to other amino
6
equimolar amounts of thiol reagents. The radical mechanism acids, which may disturb the structure and/or activity of
of TYC also makes it a robust and versatile method that can the labelled proteins. Furthermore, most of the TYC labeling
reactions were carried out in organic solvents, which limited
6
their application to native proteins. Therefore, it is necessary
a
to develop an alternative method for protein site-specific dual
labeling via the thiol–yne reaction (Scheme 1).
School of Medical Engineering, Hefei University of Technology, Hefei 230009,
P. R. China. E-mail: ymli@hfut.edu.cn; Tel: +86 0551-2904353
b
Department of Chemistry, University of Science and Technology of China, Hefei
To achieve the above idea of site-specific TYC-based protein
dual labeling, we used a pyrrolysyl-tRNA synthetase (PylRS)–
2
30026, P. R. China
c
Department of Chemistry, Tsinghua University, Beijing 100084, China
Pyl
tRNACUA pair, encoding the 22nd naturally occurring amino
†
Electronic supplementary information (ESI) available: Details of ligand syn-
thesis, photophysical measurements and cell imaging; NMR, crystallographic
data. See DOI: 10.1039/c3ob27116a
acid-pyrrolysine in response to an amber codon in archaea
1
2
species, for incorporation of alkynyl-L-lysines into proteins.
2
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the synthesis of compound 3, 4-pentyn-1-ol (840 mg, 10 mmol)
was added to a solution of triphosgene (2.9 g, 10 mmol) in dry
THF (40 mL), stirred under an ice bath for 12 h, then the
solvent was evaporated under vacuum. The collected chloro-
formate was dissolved in 5 mL THF and slowly added to a solu-
tion of Boc-Lys-OH (3.0 g, 12.2 mmol) in 1 M NaOH (25 mL)/
THF (25 mL) solution in an ice bath, the reaction mixture was
stirred at room temperature overnight. The solution was
cooled to 0 °C, washed with ice-cold Et
acidified with ice-cold 1 M HCl, and was extracted with EtOAc
2 × 30 mL). The combined organic layer was dried over
Na SO , filtered and evaporated to give a white foam. The
foam was dissolved in dry DCM (5 mL) and TFA (5 mL) was
added slowly, the reaction mixture was stirred at room temp-
2
O (2 × 25 mL) and
Scheme 1 Thiol–yne reaction mediated dual labeling of proteins. (a) Thiol–yne
radical reaction; (b) site-specific thiol–yne labeling of proteins carrying an alkyne
handle via genetically encoded alkynyl-pyrrolysine analogues.
(
2
4
The incorporation of an alkyne containing amino acid into erature for 1 h, the solvent was evaporated under vacuum, the
proteins provides the ideal labeling site for the thiol–yne reac- product was precipitated with Et O, and the precipitate was
2
tion. Note that the PylRS–tRNA pair has recently been adapted collected and dried under vacuum, affording pure 3 (0.8 g,
for incorporation of a variety of UAAs into proteins in bacteria, overall yield 31%). Compounds 1 (0.9 g, overall yield 37%) and
1
3
mammalian cells and living animals. Inspired by our pre- 2 (0.8 g, overall yield 35%) were synthesized following similar
viously evolved ACPK-RS that can work in both bacterial and synthetic procedures using 3-butyn-1-ol (700 mg, 10 mmol)
1
4
mammalian species with high incorporation efficiency, we and propargyl alcohol (560 mg, 10 mmol) as starting materials
designed and synthesized three alkyne-containing pyrrolysine respectively.
analogues and tested whether or not they can be recognized by
With compounds 1–3 in hand, we then compared the incor-
ACPK-RS. After the incorporation was successful, we then uti- poration efficiency and fidelity of ACPK-RS on these com-
lized the ACPK-RS–tRNA pair to introduce compound 3 into an pounds in E. coli. Protein expression was carried out in BL21-
E. coli acid-chaperone HdeA. This site-specifically incorporated DE3 cells co-transformed with plasmids expressing the
Pyl
CUA
alkyne handle was subjected to a thiol–yne reaction with a ACPK-RS–tRNA
pair and previously optimized HdeA-
thiol-containing N,N′-bis(dansyl) cystamine. To our best knowl- V58TAG plasmid. It has been reported that HdeA is an essen-
edge, this study presents the first thiol–yne mediated protein tial acid-chaperone in supporting the acid-resistance of E. coli
dual labeling via genetic incorporation of an alkynyl-L-lysine cells and it was suitable for being used as our model protein
1
5
derivative as a general bioorthogonal handle.
in this study.
Cells were grown in an LB medium
with shaking overnight at 37 °C. After 1 : 100 dilution in LB
medium, the culture was grown at 37 °C to an OD600 of ∼0.6.
Compounds 1–3 (100 mM for stock solution) were diluted by a
factor of 1 : 100 to a final concentration of 1 mM and incu-
bated with cell culture for 30 min. Protein expression was
induced by the addition of arabinose and IPTG to the final
concentration of 0.2% and 0.5 mM at 30 °C, respectively. After
expression for 12–14 h, cells were harvested by centrifugation
and re-suspended in lysis buffer. Bacterial lysate after soni-
cation was further purified by a Ni-NTA column. Eluted pro-
teins from a Ni-NTA column were analyzed by SDS-PAGE and
ESI-MS. Fig. 1a indicates that HdeA-V58-3 shows the best
expression level, which was only slightly lower than wild type
Results and discussion
First, we synthesized three alkyne-containing pyrrolysine ana-
logue compounds 1–3 (Scheme 2). To provide an example for
(
WT) HdeA protein. The yield of HdeA-V58-3 was estimated to
−
1
be nearly 20 mg L . We further confirmed the purity of
expressed HdeA-V58-3 by ESI-MS analysis (Fig. 1b).
We next performed site-specific radical thiol–yne coupling
on HdeA with N,N′-bis(dansyl) cystamine after confirming
the feasibility of obtaining a high yield of HdeA-V58-3.
8
Fluorophores were synthesized as previously reported. In
theory, the protein should be labelled with two N,N′-bis-
(
dansyl) cystamines via two thiols containing compounds
3
c
coupling to one alkyne handle. Similar to our recently
reported thiol–ene reaction, about 1 mM HdeA-V58-3 was dis-
solved in photo-irradiation buffer (0.2 M acetate buffer pH = 4
Scheme 2 Synthesis of alkyne-containing pyrrolysine analogues 1–3.
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Fig. 2 Dual labeling of alkyne-bearing HdeA protein via a thiol–yne reaction.
(
a) The structure of N,N’-bis(dansyl) cystamine; (b) fluorescent labeling of
HdeA-V58-3 with N,N’-bis(dansyl) cystamine by photo-irradiation for 15 min (ii),
.5 h (iii) and 2 h (iv), purified WT HdeA (i) was used as a control; (c) ESI-MS
analysis of HdeA-V58-3 dual labeling with fluorophores by a thiol–yne reaction
m/z calculated: 13 384.8 Da, found: 13 383.7 Da). Note that the mass spec-
Fig. 1 Genetic incorporation of alkyne-bearing pyrrolysine analogues (1–3)
with different side-chain lengths into proteins by an ACPK-RS–tRNA pair. (a)
SDS-PAGE analysis demonstrating the incorporation efficiencies of compounds
0
(
1–3 into HdeA protein in E. coli; (b) ESI-MS analysis of the full length of HdeA-
trometry data show a small fraction of different products besides dual labelled
HdeA-N,N’-bis(dansyl) cystamine, we consider that the different products are
the by-products of long period UV-irradiation. Admittedly, it was difficult to
determine the structure of products by ESI-MS analysis.
myc-His protein containing 3 at residue V58 (m/z calculated: 12 768.5 Da,
6
found: 12 767.8 Da).
or 0.2 M phosphate buffer pH = 6 or 7, the scale of reactions is
about 300–500 μl) and reacted with a 5–10 fold amount of
fluorophore via 365 nm UV light irradiation with 15 mM
reduced glutathione. We first performed photo-irradiation by
UV light (365 nm) using a CL-1000 ultraviolet cross-linker
(UVP) installed with 365 nm wavelength UV lamps (80–100 V)
at a distance of 3 cm at 25 °C. However, the labeling efficiency
was relatively low after 2 h of irradiation. To increase the
efficiency, we then used a high powered UV lamp (250 V
mercury lamp) in front of a 365 nm optical filter and samples
were placed in a cuvette cell at a distance of 1 cm at 25 °C for
photo-irradiation. During the reaction, a photo-induced cata-
lyst, such as 5 mM VA-044 or 2,2-dimethoxy-2-phenylaceto-
phenone (DPAP, 10%), is necessary for the reaction. After
irradiation, excess fluorophores were removed by passing
through a desalting column followed by buffer exchange to
NTA buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM DTT,
pH 7.4). We used fluorescence SDS-PAGE gel to monitor the
progress of the reaction. Fig. 2b clearly shows that alkyne con-
taining protein was successfully labelled with N,N′-bis(dansyl)
cystamine in the first 15 min and the labeling efficiency
increased obviously after two hours of irradiation. Meanwhile,
Fig. 3 (a) Native PAGE gel confirming the formation of WT HdeA and dual
labelled HdeA-N,N’-bis(dansyl) cystamine; (b) CD spectrum analysis of the struc-
ture of WT HdeA and HdeA-N,N’-bis(dansyl) cystamine.
wild type HdeA protein was used as a control to make a fair shows that fluorescent labelled N,N′-bis(dansyl) cystamine
comparison and failed to react with fluorophores under identi- forms a dimer at pH 7.0 in the same way as recombinant
cal reaction conditions. ESI-MS analysis further verified that HdeA. Furthermore, the CD spectra of both recombinant HdeA
HdeA-V58-3 was labelled with two N,N′-bis(dansyl) cystamines and fluorescence labelled HdeA superimpose well (Fig. 3), indi-
via a thiol–yne radical reaction only with a small fraction of cating that they fold to a similar three-dimensional structure.
by-products possibly induced by UV-irradiation. According to Thus, we concluded that the presence of an organic dye and a
the peak intensity of products, we estimated that the yield of radical initiator or a long period of 365 nm UV irradiation did
this reaction is about 60%.
not disturb the structure of the folded HdeA protein.
To determine whether or not the photo-irradiation would
Finally, we examined the biological activity of the dual
disturb the structure of model protein, we tested the properties labelled HdeA-N,N′-bis(dansyl) cystamine by a well established
of HdeA by CD and native gel. During the reaction process, we SurA protein assay. SurA has recently been identified as an
first noted that the protein remained soluble, suggesting that in vivo client protein of HdeA. We evaluated the protein–
no denaturation occurred. Moreover, the native PAGE analysis protein interaction by measuring SurA’s aggregation propensity
2
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Experimental section
General information
All reagents and solvents were purchased from Sinopharm
Chemical Reagent Co. Ltd or Alfa Aesar and were purified
when necessary. All other commercially available reagents and
solvents were used as received without further purification
unless otherwise indicated. Analytical HPLC was run on a
SHIMADZU (Prominence LC-20AT) instrument using an
analytical column (Grace Vydac “Protein C18”, 250 × 4.6 mM,
μm particle size, flow rate 1.0 mL min− , rt). Analytical injec-
1
5
tions were monitored at 214 and 254 nm. Semi-preparative
HPLC was run on a SHIMADZU (Prominence LC-20AT) instru-
ment using a semi-preparative column (Grace Vydac “Peptide
C18”, 250 × 10 mM, 10 μm particle size, flow rate 3.0 mL
−
1
Fig. 4 HdeA suppresses the aggregation of its client protein-SurA at a low pH min ). MALDI-TOF mass spectra were measured on an
(
a) and after the pH neutralization (b) SDS-PAGE analysis was followed by
coomassie blue staining of the soluble supernatant (S) and aggregated pellet
P) protein in each sample.
Applied Biosystems 4700 Proteomics Analyzer 283. High-
resolution ESI mass spectra were measured on a Bruker
APEX IV Fourier Transform Ion Cyclotron Resonance Mass
spectrometer. Normal ESI mass spectra were measured on a
(
1
13
Bruker Daltonics Data Analysis 3.0 workstation. H and
in the presence and absence of HdeA-N,N′-bis(dansyl) cysta- NMR spectra were recorded on an Oxford 300 MHz spectro-
mine. As shown in Fig. 4, a low pH value (pH = 2) caused SurA meter in deuteriochloroform (CDCl ) or deuterium oxide (D O)
to fully aggregate in the absence of dual labelled HdeA. with the solvent residual peak (CDCl
C
3
2
1
3
: 7.26 ppm ( H),
2
O: 4.79 ppm ( H)) as an internal reference
1
3
1
However, a significant fraction of SurA became soluble both at 77.23 ppm ( H), D
pH = 2 and after pH neutralization when dual-labelled HdeA unless otherwise stated. Data are reported in the following
was added. Because wild type HdeA proteins have been order: chemical shifts are given (δ); multiplicities are indicated
8
,14b
reported to carry out the same bio-function,
cluded that the photo-irradiation did not disturb the biological q (quartet), m (multiplet), app (apparent); coupling constants,
we then con- as br (broadened), s (singlet), d (doublet), t (triplet),
activity of model protein HdeA.
J, are reported (Hz); integration is provided. For molecular
biology and biochemistry experiments, primers were ordered
from Sangon Biotech (Shanghai) Co., Ltd. Enzymes were
ordered from New England Biolabs (NEB). Bacterial cells were
grown in LB (Luria–Bertani) broth or on LB agar medium
Conclusions
In summary, three alkynyl-pyrrolysine analogues were success- (Sigma). Antibiotics were used at final concentrations of
fully incorporated into a protein with high yields, especially 100 μg mL− for ampicillin and kanamycin and at 50 μg mL
1
−1
−1
for compound 3 (estimated yield = 20 mg mL ). Then, the for chloramphenicol (Sigma). All pictures of protein gels
alkyne-containing proteins were site-specifically dual-labelled including coomassie stained SDS-PAGE gel and native PAGE
with thiol containing fluorophores under 365 nm UV light gel were taken on ChemDocXRS+ (Bio-Rad).
irradiation. This metal-free radical induced thiol–yne coupling
Protein expression and purification
provides a promising strategy for protein dual labeling and
modification. Moreover, the structure and biological activity In detail, HdeA protein expression was carried out in E. coli
assay demonstrated that the labelled protein possesses a DH10B cells co-transformed with plasmids expressing the
Pyl
similar enzymatic activity and conformation to the wild type PylRS–tRNACUA pair and HdeA-V58TAG. Cells were grown in LB
−
1
HdeA. In current work, fluorophores were used as model com- medium containing ampicillin (100 μg mL ) and chloramphe-
pounds to confirm that site-specific dual labeling of proteins nicol (50 μg mL− ) with shaking overnight at 37 °C. After
can be achieved via a thiol–yne ligation reaction. In future 1 : 100 dilution in LB medium containing ampicillin (100 μg
1
−
1
−1
plans, this dual labeling strategy should be expanded to other mL ) and chloramphenicol (50 μg mL ), the culture was
protein modification, such as glycosylation. It has been grown at 37 °C to an OD600 of ∼0.5. Compounds 1–3 (100 mM
reported that the peptide/protein double glycosylation can for stock solution) were diluted by a factor of 1 : 100 to a final
affect much more substantially than monoglycosylation the concentration of 1 mM and incubated with cell culture for
9
,10
peptide/protein structure and biological activity.
Taken 30 min. Protein expression was induced by the addition of
together, we conclude that this genetic-code expansion arabinose and IPTG to the final concentration of 0.2% and
method in conjunction with the thiol–yne labeling may 0.5 mM, respectively. After expression for 12 h, cells were
become a complementary approach to the growing arsenal of harvested by centrifugation (10 000 g, 10 min), and resus-
strategies for protein bioorthogonal ligation.
pended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl,
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pH 7.0). Bacterial lysate after sonication was loaded onto a
Ni-NTA column (Histrap 5 mL, GE healthcare). The column
Acknowledgements
was washed with 30 mL washing buffer (20 mM Tris-HCl, This work was supported by the National Natural Science
00 mM NaCl, pH 7.0 with 40 mM imidazole) and then eluted Foundation of China (21102083 to Y. M. L.; 20932006 and
with elution buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.0 21272223 to Q. X. G.). We thank Prof. Peng R. Chen for the
3
with 250 mM imidazole).
aaRS/tRNA plasmids.
Proteins fluorescent labeling by thiol–yne reaction
The reaction was performed in the 0.2 M acetate buffer (pH =
4
.0) or 0.2 M phosphate buffer (pH = 6 or 7). The final concen-
Notes and references
trations of the reactants were as follows: HdeA-V58-3: 1 mM;
N,N′-bis(dansyl) cystamine: 20 mM. VA-044: 5 mM or 2,2-
dimethoxy-2-phenylacetophenone (DPAP, 10%) as a radical
initiator; reduced glutathione: 15 mM. The scale of this photo-
irradiation reaction is about 300–500 μl. Photo-irradiation was
performed by a high powered UV lamp (250 V mercury lamp)
in front of a 365 nm optical filter and samples were placed in a
cuvette cell at a distance of 1 cm at 25 °C. After irradiation,
excess fluorophores were removed by passing through a desalt-
ing column and the buffer was exchanged to NTA buffer
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For SDS-PAGE, samples were loaded onto 12% SDS-PAGE gels
and electrophoresed for 30 min at 80 V and 50 min at
1
50 V. The native PAGE gel was prepared using a Bio-Rad Mini-
PROTEIN Tetra Electrophoresis System. SDS was removed from
the ingredients of both the stacking gel (pH 6.8, 4%) and the
resolving gel (pH 8.8, 15%). All the protein samples were pre-
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running buffer (1 L) consists of 14.4 g glycine and 3.03 g Tris
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2
conditions (150 V, 400 mA, 60 min). A soybean trypsin inhibi-
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∼
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−
1
concentration of 0.4 mg mL . The result was recorded by
averaging three scans and plotted as mean residue ellipticity
2
−1
[
θ] (mdeg cm dmol ).
SurA assay
5
SurA protein was incubated at a concentration of 8 μM in
buffer A (8 mM H
3 4 4 2 4
PO , 150 mM KCl, and 150 mM (NH ) SO ,
pH 2.0) at 37 °C for 30 min in the absence or presence of
3
0
2 μM HdeA. After 30 min, the pH was neutralized by adding
.133 volumes of buffer B (0.5 M sodium phosphate, pH 8).
After 30 min at 37 °C, the soluble supernatant (S) and aggre-
gated pellet (P) protein in each sample at the indicated HdeA :
SurA ratios were separated by spinning at 12 000 g at 4 °C
using a SIGMA 3–18 K centrifuge, and then analyzed by
SDS-PAGE.
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