A genetically encoded acrylamide functionality

Article abstract of DOI:10.1021/cb400267m

N^ε-Acryloyl-l-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination with tRNA_CUA ^Pyl. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with N^ε-acryloyl-l-lysine is efficiently modified with thiol-containing nucleophiles at slightly alkali conditions, and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with N^ε-acryloyl-l-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.

Full text of DOI:10.1021/cb400267m

Letters
pubs.acs.org/acschemicalbiology
A Genetically Encoded Acrylamide Functionality
Yan-Jiun Lee, Bo Wu, Jeffrey E. Raymond, Yu Zeng, Xinqiang Fang, Karen L. Wooley,
and Wenshe R. Liu*
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
S
* Supporting Information
ABSTRACT: Nε-Acryloyl-L-lysine, a noncanonical amino acid with an electron deficient olefin, is
genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination
with tRNAPCyUl A. The acrylamide moiety is stable in cells, whereas it is active enough to perform a
diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-
addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein
incorporated with Nε-acryloyl-L-lysine is efficiently modified with thiol-containing nucleophiles at
slightly alkali conditions, and the acrylamide moiety also allows rapid radical copolymerization of
the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions,
the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile
imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific
fluorescent labeling of proteins incorporated with Nε-acryloyl-L-lysine both in vitro and in living
cells. This critical development allows easy access to an array of modified proteins for applications
where high specificity and reaction efficiency are needed.
elective labeling has its great utility as a tool to interrogate
physiological functions of proteins and develop biotechno-
genetically encoded in E. coli using a mutant pyrrolysyl-tRNA
S
synthetase (PylRS)-tRNAPCyUl A pair.22 A photoclick reaction that
exploits the nitrile imine−alkene cycloaddition reaction was
applied to fluorescently label proteins with O-allyl-L-tyrosine
incorporated in E. coli, albeit the labeling efficiency was low.23
Five other aliphatic olefin-containing NAAs have also been
encoded in Saccharomyces cerevisiae and used to undergo olefin
metathesis on proteins.24 In general, these aliphatic olefin-
containing NAAs have low reactivity toward nitrile imine and
are not capable of performing 1,4-addition under physiological
logical applications. To label a protein of interest site-
specifically, the genetic noncanonical amino acid (NAA)
incorporation approach has drawn great attention due to its
potentially broad applications.1,2 Many NAAs have been
genetically incorporated into proteins in bacteria, yeast,
mammalian cells, and even animals.3−8 Of these NAAs, a few
bioorthogonal functionalities are available, including ketone,
alkyne, and azide that undergo biocompatible reactions such as
hydrazone/oxime formation and azide−alkyne cycloaddition
reactions for site-selective protein modifications.9−18 Although
ketone, alkyne, and azide are involved in diverse organic
reaction types beyond the aforementioned bioorthogonal
reactions, most of these reactions either cannot be realized in
physiological conditions or lack selectivity in the biological
system. A genetically encoded and simple chemical moiety that
undergoes multiple types of biocompatible and selective
reactions could have broad implications for selective labeling
strategies in vitro and in vivo. One functional group that shows
many facets of organic reactivity is the carbon−carbon double
bond. Given the versatility in organic transformation, olefins
have found extensive application in synthetic organic chemistry.
Various biocompatible olefin reactions have been demonstrated
elsewhere. These include 1,4-addition, olefin metathesis, thiol−
ene click, Diels−Alder, and 1,3-dipolar cycloaddition reac-
tions.19,20 The olefin functionality has been genetically
incorporated into proteins. Using an evolved amber-suppress-
ing aminoacyl-tRNA synthetase-tRNA pair that was derived
from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase-
tRNATyr pair, Zhang et al. were able to incorporate O-allyl-L-
tyrosine into proteins in E. coli.21 The same NAA and two other
aliphatic olefin-containing tyrosine analogues were also
conditions. Recently, several groups have used wild-type and
Pyl
CUA
evolved PylRS-tRNA
pairs to site-specifically install NAAs
with a strain-promoted alkene into proteins.25−30 Reacting
rapidly with tetrazine or a tetrazole photocleavage product,
these NAAs contain a norbornene, transcyclooctene, or
cyclopropene moiety that is a relatively large functional
group. These NAAs may not be optimal options when minimal
structural perturbation of proteins is necessary. Acrylamide is a
small electron deficient olefin that undergoes several potentially
biocompatible reactions, e.g., those shown in Scheme 1. Here,
we wish to report its genetic incorporation and its mediated
diverse reactions for protein modifications. We envision that
the acrylamide moiety would serve as a user-defined multi-
functional chemical handle for different purposes of applica-
tions.
Because of the electrophilic nature of acrylamide, one may
suspect it undergoes 1,4-addition with thiol nucleophiles such
as intracellular glutathione. The kinetics of 1,4-addition of β-
mercaptoethanol (β-ME) to acrylamide was investigated. β-ME
Received: April 18, 2013
Accepted: June 4, 2013
Published: June 4, 2013
© 2013 American Chemical Society
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Scheme 1
instead of glutathione was used in our study due to the simple
NMR spectrum of its 1,4-addition product with acrylamide. At
pH 7.4, the second order rate constant determined was 0.0040
0.0003 M−1 s−1 (Supplementary Figure 1) that is twice a
reported rate constant (0.002 M−1 s−1) of the reaction between
glutathione and acrylamide at the same pH.31 Providing the
intracellular reduced glutathione concentration at 5 mM,32
acrylamide will have at least a half-life of 10 h that is ample for
protein expression. A parallel kinetic analysis at pH 8.0 inferred
lysine (BuK), were also synthesized and used during the initial
Pyl
search of mutant PylRS-tRNA
pairs. Because of the
CUA
structural similarity between Nε-acetyl-L-lysine (AcK), PrK
and AcrK, MmAcKRS1 that was evolved from Methanosarcina
mazei PylRS for the AcK incorporation also recognizes PrK and
AcrK.33 To improve its efficiency, we constructed a single-site
MmAcKRS1 library that randomized Y384. Mutations at Y384
have been shown in mutant PylRS variants for other lysine
derivatives.34,35 Clones from this library were screened against
PrK using a protocol described previously.36 Most of the
positive clones converged to a same MmAcKRS1 variant that
a second order rate constant of 0.013
0.001 M−1 s−1
(Supplementary Figure 2), indicating a linear dependence of
the logarithm of the rate constant on pH. Therefore, an
acrylamide-containing protein could be produced in cells and
then modified at slightly alkali conditions in vitro. Encouraged
by the initial kinetic studies, we synthesized Nε-acryloyl-L-lysine
has the Y384W mutation and is coined as PrKRS. The
Pyl
CUA
following testing with AcrK indicated that the PrKRS-tRNA
pair also mediates a high-level incorporation of AcrK at an
amber codon (Supplementary Figure 3). It was demonstrated
previously that wild-type PylRS recognizes CrtK albeit at a low
level.37,38 To improve the binding, a single site PylRS library
with randomization at Y384 was constructed. Clones of this
library were screened against BuK. Most positive clones
converged to a single PylRS variant that has the Y384W
mutation and is defined as BuKRS. BuKRS also recognizes
CrtK (Supplementary Figure 3).
(AcrK) and Nε-crotonyl-L-lysine (CrtK) whose structures are
Pyl
CUA
shown in Figure 1A and then searched mutant PylRS-tRNA
pairs for their genetic encoding in E. coli. Two more stable
analogous NAAs, Nε-propionyl-L-lysine (PrK) and Nε-butyryl-L-
For further characterization of the genetic incorporation of
AcrK and CrtK, PrKRS and BuKRS genes were cloned
separately into an optimized pEVOL vector under control of
an araBAD promoter to afford plasmids pEVOL-PrKRS and
Pyl
CUA
pEVOL-BuKRS that also contain a tRNA
gene under
control of the proK promoter. Transforming BL21(DE3) cells
that contained a reporter plasmid pET-sfGFP2TAG with
pEVOL-PrKRS or pEVOL-BuKRS afforded cell strains for
further protein expression. pET-sfGFP2TAG has a His-tagged
superfolder green fluorescent protein (sfGFP) gene that
contains an amber mutation at its S2 position and is under
control of a IPTG-inducible T7 promoter.22 Growing cells and
inducing with the addition of 0.2% arabinose, 1 mM IPTG, and
2 mM AcrK or CrtK resulted in the overexpression of sfGFP.
When AcrK or CrtK is absent, only a very small amount of
sfGFP could be observed (Figure 1B). Purified proteins were
then subjected to electrospray ionization mass spectrometry
(ESI-MS) analysis that showed the expected molecular weights
(for AcrK, calculated, 27,764 Da; detected, 27,762 Da; for
CrtK, calculated, 27,778 Da; detected, 27,778 Da). Although
proteins were expressed for 8 h at 37 °C, there was no apparent
1,4-addition product that could be detected by ESI-MS,
indicating intracellular stability for both AcrK and CrtK (Figure
1C).
To demonstrate 1,4-addition for protein labeling, we chose
to work with sfGFP that had AcrK incorporated at S2 (sfGFP-
S2AcrK), due to the known steric effect of the methyl group to
Figure 1. (A) Structures of AcK, PrK, AcrK, BuK, and CrtK. (B) The
site-selective incorporation of AcrK and CrtK at the S2 position of
sfGFP. (C) ESI-MS of the purified proteins.
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hinder the addition of a thiol nucleophile to CrtK. sfGFP-
S2AcrK (39 μM) was incubated with β-ME (40 mM) at 37 °C
overnight. pH 8.8 was used to elevate the reaction rate. The
reaction proceeded smoothly with little original sfGFP-S2AcrK
left after an overnight incubation (Supplementary Figure 4).
Besides the expected β-ME addition adduct, the ESI-MS
spectrum also displayed two additional peaks that are 76 and
151 Da greater than the expected product, indicating that one
and two additional β-ME molecules were covalently linked to
sfGFP-S2AcrK. Since the β-ME adducts of proteins through the
disulfide bond linkage were observed previously33 and sfGFP-
S2AcrK has two cysteine residues, these two additional peaks
clearly resulted from the disulfide covalent linkage of β-ME to
sfGFP-S2AcrK. These extra β-ME additions could be removed
by further treatment with 5 mM dithiothreitol (DTT) for 1 h
(Supplementary Figure 4). A similar reaction was carried out to
covalently link sfGFP-S2AcrK to methoxypolyethylene glycol
thiol 5 kDa (mPEGSH5k). sfGFP-S2AcrK (50 μM) was
incubated with mPEGSH5k (40 mM) at pH 8.8, 37 °C for 8 h,
and then treated with DTT (5 mM) for an additional hour. The
following SDS-PAGE analysis showed two protein bands, with
one around 5 kDa larger than the other. The relative intensities
of the two protein bands indicated roughly 50% conversion
(Figure 2). The same reaction with wild-type (WT) sfGFP did
not lead to a modified protein with a significant signal that
could be detected by SDS-PAGE.
Figure 3. Native PAGE analysis of coimmobilized sfGFP-S2AcrK in an
acrylamide hydrogel. WT sfGFP was loaded to the first lane to serve as
the standard.
systematic kinetic investigation of the olefin reaction with diaryl
nitrile imine is absent. Compound 1 was synthesized according
to the literature procedures. Reactions of 1 (5 μM) with
acrylamide were examined by detecting the fluorescent
emission from the product. The time-dependent fluorescent
emission increase at different acrylamide concentrations is
presented in Figure 4A. They all show single exponential
curvatures. The determined apparent reaction rate constants
have a linear dependence on the acrylamide concentrations
with a significant y-axis intercept (Figure 4B). The reactions
between 1 and other olefins shown in Figure 4C all display
equivalent kinetic features (Supplementary Figures 5−9).
Figure 4A also shows very different fluorescent intensities for
the final products at different acrylamide concentrations. All
these observations indicate that 2 underwent two parallel
reactions in an aqueous buffer, one with olefin and the other
with water (Figure 4C).41 On the basis of the mechanism in
Figure 4C, the pyrazoline formation follows the kinetic eq 1
Figure 2. SDS-PAGE analysis of WT sfGFP and sfGFP-S2AcrK before
and after their reactions with mPEGSH5k.
k1[O][N1]
k1[O] + k2
(1 − e−(k [O]+k )t
)
1
2
p =
Radical polymerization has been applied to undergo selective
protein modifications in physiological conditions.39,40 One
typical chemical moiety for radical polymerization is acrylamide.
To demonstrate that the acrylamide moiety in sfGFP-S2AcrK
can serve as a monomer for radical polymerization, a copolymer
hydrogel was prepared by mixing sfGFP-S2AcrK (110 μM),
acrylamide (15%), and bis-acrylamide (0.5%) and initiating
polymerization with the addition of tetramethylethylenedi-
amine (0.3%) and ammonium persulfate (3%). The reaction
solution was loaded to a well of a native PAGE gel and allowed
to solidify. By this process, sfGFP-S2AcrK was covalently
immobilized in the hydrogel and did not migrate during the
following electrophoresis process (Figure 3). However, a
similar test with WT sfGFP showed that the majority of the
protein did not form covalent linkages with hydrogel.
Moreover, as indicated by the fluorescence maintenance of
the immobilized sfGFP-S2AcrK, this radical copolymerization
reaction is compatible with preservation of the native protein
state of sfGFP.
(1)
where k1 is the second order rate constant of the reaction
between 2 and an olefin, k2 the hydrolysis rate constant of 2,
[O] the olefin concentration, and [NI] the concentration of 2.
Using this equation to analyze all the data resulted in k1 and k2
for various olefins shown in the table in Figure 4C. All
determined k2 values are similar, supporting the parallel
reaction mechanism. The results also indicated that acrylamide
reacted with diaryl nitrile imine much faster than with all the
other tested olefins, specifically 3 times faster than norbornene.
This observation is consistent with a previous report.42 The
hydrolysis of 2 was also analyzed by examining the UV
absorbance decay of 2 at 315 nm in a buffer that contained
acetonitrile and PBS with a ratio of 1:1 (Supplementary Figure
10) and gave a first order rate constant of 0.00034 0.00001
s−1, similar to the k2 values shown in Figure 4C.
To demonstrate the nitrile imine-olefin cycloaddition for the
fluorescent protein labeling, we expressed sfGFP variants that
were incorporated with AcrK, CrtK, and three other olefin-
containing tyrosine analogues, illustrated in Figure 5A as 3−5,
at the sfGFP S2 position. The genetic incorporation of 3 and 4
was demonstrated previously using a rationally designed PylRS
mutant.22 This same mutant also recognizes 5. sfGFP variants
(14 μM) were incubated with 1 (70 μM) in a Tris-HCl buffer
(pH 7.4) at RT for 1 h, fully denatured, and then analyzed by
It was demonstrated previously that olefin reacts with diaryl
nitrile imine to generate a fluorescent pyrazoline at an
appreciable rate in mild aqueous conditions.41 Using a
hydrazonoyl chloride 1 as a precursor that undergoes rapid
dissociation in water to form a diaryl nitrile imine (2), Kaya et
al. demonstrated that a genetically encoded norbornene moiety
could be used to fluorescently label proteins.29 However, a
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Figure 4. (A) Pyrazoline formation in reactions between 5 μM 1 and varying concentrations of acrylamide. Reactions were monitored by
fluorescence emission at 480 nm, with an excitation light at 318 nm. (B) Linear dependence of apparent rates of the reaction of eq 1 with acrylamide
on the acrylamide concentrations. (C) Mechanism of the reaction between 1 and olefin in aqueous conditions and the finally determined rate
constants for different olefins.
Figure 5. (A) Fluorescent labeling of sfGFP variants with 1. The image at the top panel was the Coomassie blue stained gel; the bottom panel was
the fluorescent image of the same gel before Coomassie blue staining. The image was detected by the BioRad ChemiDoc XRS+ system under UV
irradiation. (B) ESI-MS spectra of the original (in red) and labeled (in blue) sfGFP-S2AcrK proteins. Calculated molecular weight of the labeled
protein is 28,083 Da.
SDS-PAGE. The cyclization product of sfGFP-S2AcrK and
sfGFP with CrtK incorporated at S2 (sfGFP-S2CrtK) displayed
detectable fluorescent emission when irradiated by UV light
(Figure 5A). The signal from sfGFP-S2CrtK is relatively weak
and was estimated as 17% of that from sfGFP-S2AcrK. This was
consistent with the simulation results for reactions of 1 with
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Figure 6. (A) Labeling OmpX-AcrK in a cell lysate with 1. The first lane shows a control without OmpX-AcrK expressed. (B) Confocal fluorescent
imaging of cells overexpressing OmpX-AcrK and labeled with 1. The top left image shows the fluorescent imaging of multiple cells; the top right is
the corresponding DIC image; the bottom right is the DIC image of a single cell; the bottom left is the overlay of the DIC and the confocal
fluorescent image of a single E. coli.
acrylamide and crotonamide (Supplementary Figure 11).
However, sfGFP variants incorporated with 3−5 did not
show significant fluorescent signals after their reactions with 1.
Prolonging the reaction time did not appreciably improve the
labeling efficiency. Giving the low cyclization reactivity of 3−5
with 1, 1 preferentially underwent hydrolysis, leading to low
labeling efficiency (Supplementary Figure 11). To verify the
cyclization product of sfGFP-S2AcrK, it was subjected to ESI-
MS analysis. The major detected peak (28,085 Da) agrees well
with the calculated labeling product (28,083 Da) (Figure 5B).
The original protein could be barely detected.
uniformity of dye states consistent with single-site covalent
binding of the dye to the cell membrane and not of multiple
dye environments typical of electrostatically bound or
internalized dyes (Supplementary Figure 12). We also noticed
that the labeled OmpX-AcrK is not uniformly distributed on
the E. coli cell membrane. This preferential accumulation of
OmpX at the two poles of E. coli needs to be further
investigated. The same reaction to label E. coli cells without
OmpX-AcrK did not yield any fluorescent cells that could be
detected by confocal fluorescent microscopy (Supplementary
Figure 13).
To explore the possibility of labeling the acrylamide moiety
with 1 in living cells, plasmid pETDuet-OmpXTAG was
constructed for expressing the E. coli outer membrane protein
OmpX incorporated with AcrK (OmpX-AcrK). The OmpX
gene in pETDuet-OmpXTAG has an AAAAXAA (A denotes
alanine; X denotes an amber mutation) insertion between its
two extracellular OmpX residues S53 and S54. Transforming
BL21(DE3) cells with pEVOL-PrKRS and pETDuet-OmpX-
TAG and growing the transformed cells in LB medium
supplemented with 1 mM IPTG and 5 mM AcrK afforded
OmpX-AcrK overexpression (Figure 6A). Incubating the lysate
of cells expressing OmpX-AcrK with 1 (0.5 mM) at RT for 1 h
led to a detectable fluorescent band in a SDS-PAGE gel that
corresponded well with the Coomassie blue stained OmpX-
AcrK in the same gel. While many proteins in the lysate were
present at higher expression levels, in-gel fluorescence imaging
showed that only the expressed OmpX-AcrK could be
specifically labeled, giving nearly no detectable background
(Figure 6A). With the success of the cell lysate labeling,
experiments were conducted to fluorescently label living cells
with 1 by incubating the PBS-washed E. coli that overexpressed
OmpX-AcrK with 1 (0.5 mM) at RT for 1 h. The cells were
then washed with an isotonic saline solution and subjected to
confocal microscopy imaging. E. coli cells expressing OmpX-
AcrK showed detectable fluorescence (Figure 6B), indicating
the cycloaddition is biocompatible and highly selective. The low
variation in florescence lifetime profiles for all cells indicates a
In summary, we report a method for the genetic installation
of a small electron deficient olefin, acrylamide, into
recombinant proteins in E. coli. Being a multifaceted chemical
group for functionalizing proteins, the acrylamide moiety is
relatively stable in physiological conditions and undergoes a
number of biocompatible and unique reactions. We demon-
strate that acrylamide-containing AcrK can be genetically
Pyl
CUA
encoded using an evolved PrKRS-tRNA
pair and the
genetically incorporated AcrK mediated efficient 1,4-addition,
radical copolymerization, and 1,3-diplor cycloaddition reactions
on proteins. These unique reactions provide a direct route to
applications that include synthesizing PEGylated therapeutic
proteins for disease intervention and generating protein-
immobilizing hydrogels for ELISA, microarray/microfluidic
detections. One reaction is proven viable for site-selective
protein labeling in living cells for protein functional
investigations. The 1,4-addition reaction to PEGylate an
acrylamide in a protein produces a selective and simple
thioether linkage that is likely well tolerated in the biological
system, potentially providing an advantage over the existing
PEGylation techniques. The nitrile imine-acrylamide cyclo-
addition reaction presented in this study displays fast kinetics
and high selectivity and is performed without catalyst or light
activation. This reaction can be potentially used as an excellent
alternative for in vivo protein labeling to the most popular click
reaction type, the azide−alkyne cycloaddition that requires a
Cu(I) catalyst, and therefore may disrupt certain cellular
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Letters
functions.43,44 The current study also identified mutant PylRS
variants that allow the synthesis of proteins incorporated with
PrK, BuK, and CrtK. Propionylation, butylation, and
crotonylation are naturally existing posttranslational lysine
modifications of proteins.45,46 In line with others,37,38 we have
provided approaches that can be applied to investigate these
novel posttranslational modifications.
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ASSOCIATED CONTENT
■
S
* Supporting Information
NAA synthesis, plasmid constructions, clone identification,
protein expression, kinetic analysis, protein labeling, confocal
imaging, and additional ESI-MS spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(W.R.L.) E-mail: wliu@chem.tamu.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(19) Sletten, E. M., and Bertozzi, C. R. (2009) Bioorthogonal
chemistry: fishing for selectivity in a sea of functionality. Angew. Chem.,
Int. Ed. 48, 6974−6998.
This work was supported in part by the National Institutes of
Health (grant 1R01CA161158 to W.R.L.), the National Science
Foundation (grant CHEM-1148684), and the Welch Founda-
tion (grant A-1715 to W.R.L.). We also thank Dr. Y. H.
Rezenom from Laboratory for Biological Mass Spectrometry at
Texas A&M University for characterizing proteins with
electrospray ionization mass spectrometry.
(20) Li, Y. M., Yang, M. Y., Huang, Y. C., Song, X. D., Liu, L., and
Chen, P. R. (2012) Genetically encoded alkenyl-pyrrolysine analogues
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selective incorporation of alkenes into proteins in Escherichia coli.
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