Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation

Article abstract of DOI:10.1021/ja5055862

The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as

Full text of DOI:10.1021/ja5055862

Article
pubs.acs.org/JACS
Genetically Encoded Optochemical Probes for Simultaneous
Fluorescence Reporting and Light Activation of Protein Function
with Two-Photon Excitation
Ji Luo,†,‡ Rajendra Uprety,‡ Yuta Naro,† Chungjung Chou,‡ Duy P. Nguyen,§ Jason W. Chin,§
and Alexander Deiters*,†
†Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
‡Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States
§Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom
S
* Supporting Information
ABSTRACT: The site-specific incorporation of three new coumarin lysine
analogues into proteins was achieved in bacterial and mammalian cells using
an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded
coumarin lysines were successfully applied as fluorescent cellular probes for
protein localization and for the optical activation of protein function. As a
proof-of-principle, photoregulation of firefly luciferase was achieved in live
cells by caging a key lysine residue, and excellent OFF to ON light-switching
ratios were observed. Furthermore, two-photon and single-photon
optochemical control of EGFP maturation was demonstrated, enabling the
use of different, potentially orthogonal excitation wavelengths (365, 405, and
760 nm) for the sequential activation of protein function in live cells. These
results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and
regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific
incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation
wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional
tool for cellular biology.
tRNA synthetase/tRNA pairs for the incorporation of sterically
demanding amino acids17−20 prompted us to synthesize
coumarin lysines 1−3 (Figure 1A) and to test their
incorporation into proteins. The photochemical characteristics
of these UAAs complement and enhance the properties of
caged and fluorescent amino acids that have been genetically
encoded in bacterial and mammalian cells.19−25 Lysines 1−3
were assembled in three steps from their corresponding
coumarin alcohols (Supporting Information, Scheme S1).
Briefly, the coumarin alcohols were activated with nitrophenyl
chloroformate and coupled to commercially available Boc-
lysine. A global deprotection under acidic conditions furnished
the corresponding coumarin derivatives 1−3 in good yields.
All three coumarin lysines 1−3 contain identical benzopyr-
one cores as fluorescent probes. However, subtle substitutions
result in a set of coumarin derivatives with unique photo-
chemical properties. Introduction of a bromine at the 6-
position enables decaging not only with UV (single photon)
light (in case of 1), but also near IR (two-photon) excitation
(in case of 2).11 In contrast, extension of the coumarin−
carbamate linker by a single carbon atom results in coumarin
INTRODUCTION
■
Good photochemical properties, chemical stability, and ease of
synthesis make coumarins an important class of fluorescent
probes for biological studies.1−3 In addition to being versatile
fluorophores, coumarin chromophores can be used as light-
removable protecting groups, so-called “caging groups”, that are
photolyzed through one- and two-photon irradiation.4 Caged
molecules have been extensively applied in the optical control
of cellular processes.5−9 In particular, the 6-bromo-7-hydrox-
ycoumarinmethyl caging group undergoes fast two-photon
photolysis at 740 nm and has been used to optically control
neurotransmitters, secondary messengers, and oligonucleoti-
des.10−12 Two-photon irradiation enables optical activation of
biological processes with enhanced tissue penetration of up to 1
mm. Moreover, two-photon caging groups can be released with
greater precision in three-dimensional space than simple one-
photon caging groups.4,13
Here we report the site-specific incorporation of three
coumarin amino acids into proteins via genetic code expansion
with unnatural amino acids (UAAs)14−16 to integrate the
optical properties of coumarin probes into cellular systems.
Genetic code expansion requires the addition of orthogonal
translational machinery to achieve site-specific UAA incorpo-
ration into proteins. Recent advances in engineering pyrrolysyl-
Received: June 7, 2014
Published: October 23, 2014
© 2014 American Chemical Society
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Article
unnatural amino acids with new functions, including post-
translational modifications, bioconjugation handles, photo-
cross-linkers, photocaging groups, and others.14 Thus, we
generated and screened a panel of MbPylRS mutants, guided by
mutants that were previously reported,14 to direct the
incorporation of 2 in response to a TAG amber codon in
mammalian cells using a mCherry-TAG-EGFP reporter. Cells
containing a MbPylRS mutant with only two amino acid
mutations Y271A and L274M showed UAA-dependent
expression of full-length mCherry-EGFP-HA. The Y271A
mutation has previously been reported to direct the
incorporation of Nε-carbamate-linked lysines,33 while the
L274M mutation34,35 was discovered to facilitate higher
amber suppression activities with 2 in vivo, because it allows
greater flexibility of the side chain and imposes less steric bulk
at the back of the hydrophobic pocket. This synthetase, termed
BhcKRS, enabled the site-specific incorporation of not only 2
but also 1 and 3 in response to the amber codon TAG within
sfGFP-Y151TAG-His6 in E. coli (Figure 1). This is not
surprising, considering the very similar structures of 1−3 and
previous observations of the high promiscuity of PylRS.36,37 To
further rationalize the ability of BhcKRS to incorporate 1−3,
molecular modeling was employed. The wild-type PylRS
structure (PDB: 2Q7H) was used as a starting template for
which the Y271A and L274M mutations were introduced using
Modeller.38 The mutant structure was energy minimized in
Amber molecular dynamics39 before docking 1−3 into the
active site pocket using AutoDock4.40 As expected, 1−3 adopt
very similar poses, reflecting their similarity in structure (see
Supporting Information, Figure S1). The mutated synthetase
model reveals that the Y271A and L274M mutations greatly
enlarge the binding pocket to accommodate the bulky bicyclic
caging group, while also orienting it in a favorable π-stacking
interaction with W382. This orientation also benefits from a
favorable H-bond interaction between the coumarin hydroxyl
group and D373. Similar to published crystal structures, the
amino group’s positioning is maintained by interactions with a
structural water and Y349.41 It has been previously shown that
interactions with N311 and R295 play an important role in
amino acid recognition by the PylRS system.31,41,42 The docked
structure maintains these key interactions with the carbamate
carbonyl forming a H-bond with N311, while the carboxylic
acid forms a H-bond with R295 (Figure 1B,C).
Figure 1. (A) Structures of the genetically encoded coumarin amino
acids for fluorescence reporting and light activation of protein
function. (B) Crystal structure of PylRS (2Q7H) with the pyrrolysine
substrate (yellow) in the active site. (C) Structure of BhcKRS with 1
(green) docked into the active site. Dashed blue lines represent H-
bond interactions. (D) SDS-PAGE analysis of sfGFP-Y151TAG
containing 1−3 through incorporation in E. coli. The gel was stained
with Coomassie blue (top), and coumarin fluorescence was imaged via
excitation at 365 nm (bottom). (E) Fluorescence micrographs of HEK
293T cells expressing the BhcKRS/tRNACUA pair and mCherry-TAG-
EGFP-HA in the presence or absence of 1−3. (F) Western blot
analysis of cell lysates using an anti-HA antibody and a GAPDH
antibody as a loading control. Full-length protein expression is only
observed in the presence of 1−3, and incorporation efficiency with all
three amino acids is similar in mammalian cells.
SDS-PAGE analysis reveals coumarin fluorescence of the
expressed proteins containing the coumarin lysines 1−3. No
fluorescence is observed for wild-type sfGFP because its
excitation wavelength (488 nm) does not match that of 1−3
(365 nm) and because of the denaturing conditions of the gel.
The dependence of protein expression on the presence of 1−3
demonstrates that the engineered BhcKRS synthetase has a
high specificity for coumarin lysines and does not significantly
incorporate any of the common 20 amino acids. Similar results
were obtained for the incorporation of 1−3 into ubiquitin and
myoglobin in E. coli. Electrospray ionization mass spectrometry
(ESI-MS, Supporting Information, Figures S2−S4) showed that
recombinantly expressed sfGFP-1 and -3 have a mass of
28446.22 and 28460.60 Da, in agreement with the expected
masses of 28446.03 and 28460.04 Da, respectively. ESI-MS
analysis of sfGFP-2 showed a mass of 28445.97 Da, indicating a
partial loss of bromine during E. coli expression, possibly due to
reductive dehalogenation.43 Overall, these results demonstrate
that 1−3 can be incorporated into proteins in E. coli in good
lysine 3, which does not undergo photolysis and thus
represents a stable coumarin amino acid probe. Thus, coumarin
lysines 1 and 2 can be used as both fluorescent and light-
activated probes for optochemical control of protein function
using UV or near-IR light, while coumarin lysine 3 may serve as
a stable fluorescent probe that does not decage under UV
excitation.
RESULTS AND DISCUSSION
■
The Methanosarcina barkeri pyrrolysyl tRNA synthetase/
tRNACUA (MbPylRS/tRNACUA) is functional and orthogonal
in a wide range of organisms, such as Escherichia coli, yeast,
mammalian cells, and animals such as Caenorhabditis elegans
and Drosophila melanogaster.24,26−31 Furthermore, wild-type
PylRS recognizes several unnatural amino acids without
accepting any of the 20 common amino acids as a substrate.32
The active site of the PylRS can be further engineered through
directed evolution to enable the incorporation of additional
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yields (8.0 mg/L, 1.6 mg/L, and 2.5 mg/L, respectively, for
sfGFP) and with high specificity.
removed from the protein, while the continued presence of the
Coomassie-stained protein band indicates stability of the
protein. In a cellular context, this may enable experiments
that allow for the determination of protein expression, protein
localization, and protein decaging using a single optochemical
probe in a single experiment. In contrast, insertion of an extra
methylene unit between the lysine and the fluorophore fully
abrogates photocleavage and thus establishes 3 as a stable
amino acid for the site-specific fluorescent labeling of proteins.
No change in coumarin fluorescence is observed after UV
exposure of sfGFP-3 for 20 min (Figure 2C,D). Due to the
identical fluorophores in 1 and 3 and the stability of 3 to the
UV irradiation conditions, the loss of protein fluorescence in
sfGFP-1 is due to decaging and not due to photobleaching.
This is further supported by mass spectroscopic analysis of the
proteins before and after UV exposure (see Supporting
Information, Figures S2 and S4).
To demonstrate the ability of the genetically encoded
coumarin lysines to act as reporters for protein localization in
live cells, we investigated their utility as a protein nuclear
localization marker. A plasmid was constructed to express
EGFP-HA with an N-terminal NLS (nuclear localization signal,
pNLS-linker-EGFP-HA),44 which reliably localizes EGFP to the
nucleus (Supporting Information, Figure S6). A TAG amber
codon was introduced in the linker between the NLS and
EGFP, allowing for site-specific unnatural amino acid
incorporation without affecting EGFP formation or nuclear
translocation. Cells cotransfected with the pNLS-KTAG-EGFP
and BhcKRS/PyltRNACUA plasmid pair in the presence of 1
(0.25 mM) were analyzed for coumarin fluorescence (405 nm
excitation, 450−480 nm emission) and EGFP fluorescence
(488 nm excitation, 490−520 nm emission) by confocal
microscopy. The observation of complete colocalization of both
fluorophores in the nucleus (merged micrographs) demon-
strates the ability to use 1 as a reporter of protein localization
(Figure 2E and Supporting Information, movie S1 and Figure
S7).
To demonstrate that the coumarin lysines 1−3 can also be
genetically incorporated into proteins in mammalian cells,
pBhcKRS-mCherry-TAG-EGFP-HA and p4CMVE-U6-PylT
were cotransfected into human embryonic kidney (HEK)
293T cells. Cells were incubated for 24 h in the absence of any
unnatural amino acid and in the presence of 1−3 (0.25 mM).
Fluorescence imaging revealed EGFP expression only in the
presence of 1−3, indicating specific incorporation of the
coumarin lysines in response to the TAG codon, without
measurable incorporation of endogenous amino acids (Figure
1E). This was further confirmed by an anti-HA Western blot on
cell lysates from the same experiment (Figure 1F).
Furthermore, full-length mCherry-EGFP protein was immuno-
precipitated from HEK 293T cells using an immobilized
antibody against the HA-tag and mass spectrometry sequencing
confirmed that 1−3 are site-specifically incorporated into
protein in mammalian cells (Supporting Information, Figure
S5). Importantly, the presence of bromine was verified for
protein containing 2, confirming the genetic encoding of the
Bhc-caged lysine.
Because the coumarin groups on 1 and 2 are caging groups
that can be removed via light exposure, loss of their intrinsic
fluorescence can be used as an indicator of protein decaging
through UV irradiation, as shown in Figure 2A,B. This was
demonstrated through a UV exposure time-course of purified
sfGFP-1, followed by SDS-PAGE analysis. The coumarin
fluorescence intensity of sfGFP-1 gradually decreases with
extended UV exposure as more of the coumarin caging group is
To apply the coumarin lysines 1−3 in the optical control of
protein function in live cells, firefly luciferase (Fluc) was
selected as an initial target because bioluminescence measure-
ments afford low background, high sensitivity, and easy
quantification. On the basis of the Fluc crystal structure, a
critical lysine residue, K206, was identified, which is positioned
at the edge of the substrate-binding pocket (Figure 3B). It has
been proposed that this residue stabilizes and orients ATP in
the active site.45,46 The ε-amino group on K206 provides a
hydrogen-bond interaction with the γ-phosphate of ATP and
promotes the adenylation reaction with luciferin, thus being
essential for catalytic activity as shown by the dramatic decrease
in enzymatic activity displayed by the K206R mutant.45
Therefore, we hypothesized that a sterically demanding
coumarin caging group placed on K206 would prevent the
interaction with ATP and limit the overall access of the
substrates to the active site (Figure 3A). Photolysis of the
coumarin lysine would remove the caging group and produce a
native lysine residue, restoring the catalytic activity of the
enzyme (Figure 3B). A genetically encoded photocaged lysine
at K206 would enable the enhanced regulation of the catalytic
activity of firefly luciferase via light activation.
Figure 2. SDS-PAGE fluorescence analysis shows photodecaging of
sfGFP-1 while sfGFP-3 is stable to UV exposure. (A) Loss of coumarin
fluorescence after extended sfGFP-1 in-gel decaging for 0−50 min
(365 nm, transilluminator). (B) Coomassie staining reveals identical
sfGFP-1 protein amounts in all lanes. (C) No loss of coumarin
fluorescence is observed, since sfGFP-3 does not decage. (D)
Coomassie staining reveals identical sfGFP-3 protein amounts in all
lanes. (E) Nuclear colocalization of coumarin and EGFP fluorescence
in CHO K1 cells cotransfected with pNLS-TAG-EGFP-HA and the
BhcKRS/PylT pair (pBhcKRS-4PylT) in the presence of 1 (0.25
mM). A DIC image and a merged image of all three channels are
shown as well.
Site-directed mutagenesis of the corresponding K206 residue
to the amber codon (TAG) enabled incorporation of 1−3 into
firefly luciferase in mammalian cells. HEK 293T cells were
cotransfected with the mutated firefly luciferase plasmid
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Figure 3. Engineering of an optochemically controlled Photinus pyralis
firefly luciferase through unnatural amino acid mutagenesis. (A)
Caging groups at position K206 are blocking access to the binding
pocket by luciferin and ATP and are disrupting a required hydrogen
bonding network. (B) After decaging, wild-type Fluc is generated and
the substrates can now enter the active site. PDB: 2D1S. (C) Bright-
Glo luciferase assay of cells that were either kept in the dark or
irradiated (365 nm, 4 min). Chemiluminescence units were
normalized to the −UAA/−UV control. No enzymatic activity was
observed for the caged proteins, and significant increases in
luminescence were observed after photolysis of luciferase containing
1 or 2, while the K206 → 3 mutant was permanently deactivated, as
expected. Error bars represent standard deviations from three
independent experiments.
Figure 4. (A) Location of K85 (yellow) and interactions with D82,
C70, and S72 in EGFP. The chromophore is shown in magenta. (B)
Schematic of the pEGFP-K85TAG-mCherry construct and its
application in light activation studies. (C) Fluorescence imaging of
HEK 293T cells expressing EGFP-K85TAG-mCherry, 90 min after
irradiation at 365 nm (30 s, DAPI filter, 358−365 nm) in the presence
of 1 (Nikon A1R confocal microscope, 20× objective, 2-fold zoom).
(D) Normalized EGFP fluorescence as a function of time after 365 nm
light activation (error bars represent standard deviations from the
measurement of three independent cells, t1/2 = 49 min).
(pGL3-K206TAG) and the MbBhcKRS/PyltRNACUA pair
(pBhcKRS-4PylT) in the presence of 1−3 (0.25 mM). After
24 h incubation, the cells were either irradiated for 4 min (365
nm, 25 W) or kept in the dark. The incorporation of 1−3 into
Fluc caused complete inhibition of luciferase activity before UV
irradiation, as determined by a Bright-Glo luciferase assay,
comparable to the negative control (no unnatural amino acid).
After UV irradiation, 1 and 2 were decaged to produce native
lysine, resulting in the activation of firefly luciferase by 34-fold
and 31-fold, respectively (Figure 3C). As expected, 3 did not
show any activation of luciferase enzymatic activity upon
illumination, as it does not undergo decaging. Therefore, the
activity of firefly luciferase can be tightly optochemically
regulated by incorporation of a coumarin lysine residue into the
active site of the luciferase protein. Interestingly, attempts to
apply 1 and 2 at position K529, another site that can be used
for optical control of luciferase function,47 led to greatly
diminished luciferase activity, while introduction of our
previously reported o-nitrobenzyl-caged lysine24,48,49 worked
at both positions K206 and K529. Western blots confirmed that
both Fluc-K206 → 1 and Fluc-K529 → 1 were expressed at
similar levels in mammalian cells (Supporting Information,
Figures S8 and S9).
To observe the optical triggering of protein function via
decaging of 1 and 2 in real time, enhanced green fluorescent
protein (EGFP) was selected as a second target protein for
caging. EGFP consists of an 11-stranded β-barrel and a central
α-helix with the Thr65-Tyr66-Gly67 chromophore.50 The
chromophore plays a crucial role in EGFP fluorescence and
stability.51 Correctly folded EGFP is a prerequisite for mature
chromophore formation, with a number of lysine residues being
essential to its successful folding.52 Most notable is that only 1
lysine (K85) out of 20 is buried within the protein.52 K85 forms
a salt bridge with D82 and H-bonding interactions with the
backbone of C70 and S72,52 all of which are in close proximity
to the chromophore (Figure 4A). It has been shown that C70,
S72, and D82 are key residues for control of chromophore
formation and oxidation.53,54 We hypothesized that introduc-
tion of coumarin-caged lysines 1 and 2 at K85 would affect
D82, C70, and S72, interrupting the α-helix bending and thus
indirectly inhibiting chromophore maturation. To this end, we
envisioned that UV activation would yield native EGFP that
rapidly undergoes maturation. An EGFP mutant with an amber
codon at position K85 (pEGFP-K85TAG) was generated as a
fusion construct with mCherry, to provide a second reporter for
successful plasmid transfection and incorporation of 1 and 2.
HEK 293T cells were cotransfected with pEGFP-K85TAG-
mCherry and the BhcKRS/PyltRNA pair in the presence of 1
and 2 (0.25 mM). After 24 h, the cells were washed and
incubated in fresh media for 1 h. Cells expressing mCherry
were observed by fluorescence imaging to confirm that EGFP-
1/2-mCherry is generated in the presence of 1 or 2. Cells were
irradiated for 30 s at 365 nm, and fluorescence was imaged by
time-lapse microscopy. After photolysis of EGFP-1, green
fluorescence started to appear around 10 min, and over time
the fluorescence intensity gradually increased, reaching a
plateau at 120 min (Figure 4D and Supporting Information,
movie S2). A half-life of 49 min was observed, matching reports
of EGFP chromophore maturation as the rate-limiting step.55
Previous measurements of EGFP folding and maturation have
been exclusively performed in test tubes.56 No cellular studies
have been conducted, as a precise starting point for kinetic
analysis could not be provided.
Given that the coumarin lysines have relatively broad
absorption bands in the 300−420 nm range that enable
decaging at longer wavelengths, we speculated that irradiation
at 405 nm1,57 may efficiently activate 1 and 2. Thus, activation
through blue light irradiation using a standard laser-scanning
confocal microscope was tested. As expected, exposure at 405
nm induced fluorophore formation of EGFP-1 and -2 (Figure
5A,B). Because attempts to decage a previously incorporated
nitrobenzyloxycarbonyl lysine24,48,49 and nitrobenzyl tyro-
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and mammalian cells using an engineered BhcKRS synthetase
system. The genetically encoded coumarin lysines were
successfully applied as fluorescent cellular probes for protein
localization, and the small size of these coumarin lysines is
expected to minimally perturb protein structure and function,
unless they are placed at critical sites. In addition to their small
size, the spectral properties of 1−3 do not interfere with
common fluorescent proteins (e.g., EGFP). While the amino
acid 3 showed stability under irradiation conditions, the
coumarins 1 and 2 were readily decaged, generating wild-type
lysine residues. As a proof-of-principle, photoregulation of
firefly luciferase was achieved in live cells by caging a key lysine
residue, and excellent OFF to ON light-switching ratios were
observed for 1 and 2. As expected, the stable fluorescent amino
acid 3 did not undergo photolysis. Furthermore, two-photon
and single-photon optochemical control of EGFP maturation
was demonstrated, enabling the use of different, potentially
orthogonal, excitation wavelengths (365, 405, and 760 nm) for
the sequential activation of protein function in live cells. While
the caged lysine 2 could be activated using two-photon
irradiation at 760 nm, the lysine 1 was stable under these
conditions. However, decaging of 1 was readily achieved with
blue light of 405 nm, while a previously encoded o-nitrobenzyl-
caged lysine requires UV activation.24,48,49 These results
demonstrate that coumarin lysines are a new and valuable
class of optical probes that can potentially be used for the
investigation and regulation of protein structure, dynamics,
function, and localization in live cells. The small size of
coumarin, the application as both a light-activated caging group
and a fluorescent probe, and the broad range of excitation
wavelengths are advantageous over other genetically encoded
photocontrol systems and provide a unique and multifunctional
tool for cellular biology. The ability to incorporate all three
coumarin lysines with the same PylRS/tRNACUA pair further
facilitates their application.
Figure 5. Fluorescence confocal imaging of COS-7 cells expressing
EGFP-KTAG-mCherry, before and after irradiation at 405 nm (30
mW diode laser, 20% laser power, 12.6 μs dwell time, 8 cycles) in the
presence of 2 (A) or 1 (B) (Zeiss confocal LSM710 microscope, 40×
water objective). Similar light-activation experiments before and after
irradiation of HEK 293T cells at 760 nm (130 mW, 2 μm/s dwell time,
30 cycles, Olympus Fluoview FV1000 MPE, MaiTai DSBB-OL IR
pulsed laser), in the presence of 2 (C) or 1 (D), imaged with a
Olympus Fluoview1000, 40× oil objective.
sine19,58,59 at 405 nm were not successful on comparable time
scales and at comparable illumination power (data not shown),
the caged lysines 1 and 2 may enable multiwavelength
activation of proteins caged with the two different optical
probes.
Taking advantage of the two-photon decaging feature of 2,11
photocontrol of EGFP folding by two-photon activation of
EGFP-2 was performed. HEK 293T cells were cotransfected
with pEGPF-K85TAG-mCherry and pBhcKRS-4PylT in the
absence or presence of 1 and 2 (0.25 mM). After a 24 h
incubation, the cells were washed and incubated in fresh media
for 1 h and irradiated with a multiphoton laser (760 nm, 130
mW, 2 μm/s dwell time, 30 cycles, Olympus Fluoview FV1000
MPE Multiphoton laser scanning microscope FV10-ASW,
MaiTai DSBB-OL IR pulsed laser). Images were acquired
before and after two-photon irradiation using both EGFP (488
nm) and mCherry (561 nm) excitation. Gratifyingly, an EGFP
fluorescent signal was observed after photolysis of 2 at 760 nm
(Figure 5C). The cells expressing EGFP containing 1, as a
control, were also exposed to two-photon excitation (760 nm)
and imaged in the same fashion (Figure 5D); no EGFP
activation was observed. In addition to the increased three-
dimensional resolution that is provided through two-photon
excitation, effectively shifting the activation wavelength to the
near-IR will enable multiwavelength activation in conjunction
with other optically triggered biological processes, while also
preventing any overlap with established fluorescent reporter
proteins.
EXPERIMENTAL SECTION
■
Cloning. (1) Construction of pNLS-TAG-EGFP-HA: The pTAG-
EGFP-HA fragment was amplified from pmCherry-TAG-EGFP-HA
using the PCR primers G1/G2, digested with HindIII and BglII, and
ligated into pEGFP-N1 (Clontech), generating the pTAG-EGFP-HA
plamid. The pNLS PCR fragment was obtained by using primers N1/
N2 and then ligated into the HindIII and XbaI sites of pTAG-EGFP-
HA to generate the pNLS-TAG-EGFP-HA plasmid. (2) Construction
of pNLS-WT-EGFP-HA: Plasmids were obtained by converting the
TAG codon of pNLS-TAG-EGFP-HA into an AAG (Lys) codon using
primers QC1/QC2 and a QuikChange site-directed mutagenesis kit
(Agilent). (3) Construction of pBhcKRS-4PylT: The plasmid was
obtained by ligating the p4CMVE-U6-PylT fragment from pMbPylT
between the restriction sites NheI and MfeI sites of pMbBhcKRS.
Expression and Purification of Proteins in E. coli. The plasmid,
pBAD-sfGFP-Y151TAG-pylT was cotransformed with pBK-BhcKRS24
into E. coli Top10 cells. A single colony was grown in LB media
overnight, and 500 μL of the overnight culture was added to 25 mL of
LB media, supplemented with 1 mM of the designated unnatural
amino acid and 25 μg/mL of tetracycline and 50 μg/mL of kanamycin.
Cells were grown at 37 °C, 250 rpm, and protein expression was
induced with 0.1% arabinose when the OD600 reached ∼0.6. After
overnight expression at 37 °C, cells were harvested and washed by
PBS. The cell pellets were resuspended in 6 mL of phosphate lysis
buffer (50 mM, pH 8.0) and Triton X-100 (60 μL, 10%), gently mixed,
and incubated for 1 h at 4 °C. The cell mixtures were sonicated, and
the cell lysates were centrifuged at 4 °C, 13 000 g, for 10 min. The
supernatant was transferred to a 15 mL conical tube, and 100 μL of
Ni-NTA resin (Qiagen) was added. The mixture was incubated at 4 °C
for 2 h under mild shaking. The resin was then collected by
SUMMARY
■
The site-specific genetic incorporation of three new coumarin
lysine analogues 1−3 into proteins was achieved in bacterial
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centrifugation (1000g, 10 min), washed twice with 400 μL of lysis
buffer, and followed by two washes with 400 μL of wash buffer
containing 20 mM imidazole. The protein was eluted with 400 μL of
elution buffer containing 250 mM imidazole. The purified proteins
were analyzed by 10% SDS-PAGE and stained with Coomassie Blue.
Protein Analysis by ESI-MS. Two different instruments were
used: (A) Protein samples were analyzed using capillary LC ESI-TOF
MS. The protein samples were loaded onto a PRLP-S column
(Thermo Fisher 5 μm, 1000 A, 300 μm i.d. × 100 mm) on an LC
system (Ultimate 3000, Dionex, Sunnyvale, CA). The LC system was
directly coupled to an electrospray ionization time-of-flight mass
spectrometer (microTOF, BrukerDaltonics, Billerica, MA). Chromato-
graphic separation was performed at a constant flow rate of 3.5 μL/
min using a binary solvent system (solvent A: 2.5% acetonitrile and
0.1% formic acid; solvent B: 80% acetonitrile and 0.1% formic acid)
and a linear gradient program (0−5 min, 5% B; 5−10 min, 5−30% B;
10−30 min, 30−75% B; 30−35 min, 75−100% B; 35−45 min, 100−
5% B; 45−60 min, 5% B). Mass spectra were acquired in positive ion
mode over the mass range m/z 50 to 3000. ESI spectra were
deconvoluted with the MaxEnt algorithm (Data Analysis 3.3, Bruker
Daltonics, Billerica, MA), obtaining molecular ion masses with a mass
accuracy of 1−2 Da. (B) High-resolution exact mass measurement
were conducted on an Agilent Technologies (Santa Clara, CA) 6210
LC-TOF mass spectrometer. Samples were analyzed via a 1 μL flow
injection at 300 μL/min in a water:methanol mixture (25:75 v/v) with
0.1% formic acid. The mass spectrometer was operated in positive ion
mode with a capillary voltage of 4 kV, nebulizer pressure of 35 psi, and
a drying gas flow rate of 12 L/min at 350 °C. The fragmentor and
skimmer voltages were 200 and 60 V, respectively. Reference ions of
purine at m/z 121.0509 and HP-0921 at m/z 922.0098 were
simultaneously introduced via a second orthogonal sprayer and used
for internal calibration.
Coumarin Lysine Incorporation in Human Cells. Human
embryonic kidney (HEK) 293T cells were grown in DMEM
(Dulbecco’s Modified Eagle Medium, Gibco) supplemented with
10% FBS (Gibco), 1% Pen-Strep (Corning Cellgro), and 2 mM L-
glutamine (Alfa Aesar) in 96-well plates (Costar) in a humidified
atmosphere with 5% CO2 at 37 °C. HEK 293T cells were transiently
transfected with the pMbBhcKRS-mCherry-TAG-EGFP-HA and
p4CMVE-U6-PylT24 at ∼75% confluency in the presence or absence
of 1, 2, and 3 (0.25 mM) in 96-well plates. Double transfections were
performed with equal amounts of both plasmids. After an overnight
incubation at 37 °C, the cells were washed by PBS and imaged with a
Zeiss Axio Observer.Z1Microscope (10× objective). To confirm the
expression of the fusion protein and also differentiate between
expression levels, a Western blot was performed. HEK 293T cells were
cotransfected with pMbBhcKRS-mCherry-TAG-EGFP-HA and
p4CMVE-U6-PylT in the presence or absence of 1, 2, and 3 (0.25
mM) in six-well plates. After 24 h of incubation, the cells were washed
by chilled PBS, lysed in mammalian protein extraction buffer (GE
Healthcare) with complete protease inhibitor cocktail (Sigma) on ice,
and the cell lysates were cleared at 13 200 rpm centrifugation (4 °C, 20
min). The protein lysate was boiled with loading buffer and then
analyzed by 10% SDS-PAGE. After gel electrophoresis and transfer to
a PVDF membrane (GE Healthcare), the membrane was blocked in
TBS with 0.1% Tween 20 (Fisher Scientific) and 5% milk for 1 h. The
blots were probed and incubated with the primary antibody, α-HA-
probe (Y-11) rabbit polyclonal lgG (sc-805, Santa Cruz Biotech),
overnight at 4 °C, followed by a fluorescent secondary antibody, goat-
α-rabbit lgG Cy3 (GE Healthcare), for 1 h at room temperature. The
binding and washing steps were performed in TBS with 0.1% Tween
20.
to the expected molecular weight of mCherry-EGFP-HA were excised,
washed with HPLC water, and destained with 50% acetonitrile/25
mM ammonium bicarbonate until no visible staining. Gel pieces were
dehydrated with 100% acetonitrile and reduced with 10 mM
dithiothreitol at 56 °C for 1 h, followed by alkylation with 55 mM
iodoacetamide at room temperature for 45 min in the dark. Gel pieces
were then again dehydrated with 100% acetonitrile to remove excess
alkylating and reducing agents and rehydrated with 20 ng/μL trypsin/
25 mM ammonium bicarbonate and digested overnight at 37 °C. The
resultant tryptic peptides were extracted with 70% acetonitrile/5%
formic acid, speed-vac dried, and reconstituted in 18 μL of 0.1% formic
acid. Tryptic digests were analyzed by reverse-phased LC-MS/MS
using a nanoflow LC (Waters nanoACQUITY UPLC system, Waters
Corp., Milford, MA) coupled online to an LTQ/Orbitrap Velos hybrid
mass spectrometer (Thermo-Fisher, San Jose, CA). Separations were
performed using a C18 column (PicoChip column packed with 10.5
cm Reprosil C18 3 μm 120 Å chromatography media with a 75 μm ID
column and a 15 μm tip, New Objective, Inc., Woburn, MA). Mobile
phase A was 0.1% formic acid in water, and mobile phase B was 0.1%
formic acid in acetonitrile. Samples were injected onto a trap column
(nanoACQUITY UPLC trap column, Waters Corp., Milford, MA) and
washed with 1% mobile phase B at a flow rate of 5 μL/min for 3 min.
Peptides were eluted from the column using a 90 min gradient running
at 300 nL/min (5% B for 3 min, 5−36% B in 62 min, 36−95% B in 2
min, 95% B for 8 min, 95%−5% B in 1 min, 5% B for 16 min). The
LTQ/Orbitrap instrument was operated in a data-dependent MS/MS
mode in which each high resolution broad-band full MS spectra (R =
60 000 at mass to charge (m/z) 400, precursor ion selection range of
m/z 300 to 2000) was followed by 13 MS/MS scans in the linear ion
trap where the 13 most abundant peptide molecular ions dynamically
determined from the MS scan were selected for tandem MS using a
relative collision-induced dissociation (CID) energy of 35%. Dynamic
exclusion was enabled to minimize redundant selection of peptides
previously selected for CID. MS/MS spectra were searched with the
MASCOT search engine (version 2.4.0, Matrix Science Ltd.) against a
UniProt jellyfish proteome database (June 2014 release) from the
European Bioinformatics Institute (http://www.ebi.ac.uk/integr8)
combined with endogenous mCherry-EGFP fasta sequences. The
following modifications were used: static modification of cysteine
(carboxyamidomethylation, +57.0214 Da) and variable modification of
methionine (oxidation, +15.9949 Da) for all searches, variable
modifications of lysine for mCherry-EGFP-HA (1, +218.17 Da; 2,
+295.93 Da; 3, +231.03 Da). The mass tolerance was set at 20 ppm for
the precursor ions and 0.8 Da for the fragment ions. Peptide
identifications were filtered using PeptideProphet and ProteinProphet
algorithms with a protein threshold cutoff of 99% and peptide
threshold cutoff of 95% implemented in Scaffold (Proteome Software,
Portland, OR).
Expression of Caged Firefly Luciferase and Light Activation.
HEK 293T cells were cultured in DMEM (Dulbecco’s Modified Eagle
Medium, Gibco) supplemented with 10% FBS (Gibco), 1% Pen-Strep
(Gibco), and 2 mM L-glutamine (Alfa Aesar) in 96-well plates (BD
Falcon) in a humidified atmosphere with 5% CO2 at 37 °C. At 80−
90% confluency, cells seeded on plates were transfected and the
medium was changed to fresh DMEM supplemented without or with
1, 2, or 3 (0.25 mM). The plasmid pMbBhcKRS-4PylT was
constructed containing both CMV-MbBhcKRS and 4CMVE-U6-
PylT. A TAG amber stop codon was introduced at the K206 site
using primers GL1/GL2 and a QuikChange mutagenesis kit (Agilent
Technologies). A pGL3-control plasmid containing the gene encoding
P. pyralis firefly luciferase with the TAG amber mutation at residue
K206 (pGL3-K206TAG) was cotransfected into cells with the plasmid
pBhcKRS-4PylT using linear PEI according to the manufacturer’s
protocol (Millipore). After double transfection and 24 h incubation,
the medium was changed to DMEM without phenol red, and the cells
were irradiated with UV light (365 nm) for 4 min using a 365 nm UV
lamp (high performance UV transilluminator, UVP, 25 W) or kept in
the dark. Cells were lysed by addition of 100 μL of substrate solution
(Promega) in a 96-well plate (BD Falcon), and luminescence was
measured on a Synergy 4 multimode microplate reader with an
Protein Sequencing by LC-MS/MS. HEK 293T cells were
transfected with pBhcKRS-mCherry-TAG-EGFP-HA and p4CMVE-
U6-PylT in a 10 cm Petri dish and incubated with DMEM containing
1, 2, or 3 (0.25 mM) for 24 h. Cells were lysed with extraction buffer
(GE Healthcare) and the mCherry-1/2/3-EGFP-HA protein was
immunoprecipitated using the Pierce HA Tag IP/Co-IP kit (Pierce)
according to manufacturer’s protocol. The proteins were separated on
SDS-PAGE gels and stained with silver stain. Regions corresponding
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Journal of the American Chemical Society
Article
integration time of 2 s and a sensitivity of 150 or on a Tecan M1000
microplate reader with an integration time of 1 s.
Molecular Docking Experiments. The energy-minimized mutant
structure was prepared for docking with AutoDock4 by removing all
sodium ions, and all water molecules except for a single water molecule
which exists in the active site pocket of the protein. This structural
water molecule is present in all available crystal structures and plays an
important role in amino acid recognition. The receptor input file was
prepared using AutoDock Tools software.40 The side chains for residue
L274M were treated as flexible, while all other side chains were kept
rigid. The unnatural amino acid ligands were constructed using
ChemBioDraw3D, and the molecular geometry was optimized using
the MMFF94 force field.63 The ligand input files were prepared for
docking using AutoDock Tools as well. Lamarkian genetic algorithm
was used for docking with the following parameters: number of runs:
75, ga_pop_size 150, ga_num_evals 250 000 000, ga_num_genera-
tions 27 000 were set, all other parameters were kept default. Docking
results were clustered based on RMSD of each pose. Each coumarin
lysine yielded a low energy cluster with binding scores of −9.63 kJ/
mol, −6.18 kJ/mol, and −6.14 kJ/mol for 1, 2, and 3, respectively.
Visualization of Nuclear Localization through Coumarin
Lysine Incorporation. CHO K1 cells were plated into a polylysine-
coated four-well chamber slide (Lab-Tek) and, after incubation to 75%
confluency, were transfected with 1 μg of pNLS-KTAG-EGFP and
pBhcKRS-4PylT each. After 16 h incubation at 37 °C/5% CO2 in
DMEM with 10% FBS in the presence of 1 (0.25 mM), cells were
washed with DMEM without phenol red and then incubated for 2 h.
The cells were washed with PBS, fixed with 4% formaldehyde, and
stained with rhodamine−phalloidin (Life Technologies). The chamber
slide was dried in the dark overnight and cells were imaged on a Zeiss
710 confocal microscope (40× water objective).
One-Photon Light Activation of EGFP. HEK 293T cells were
plated into a poly-D-lysine-coated eight-well chamber slide (Lab-Tek).
After incubation to 70% confluency, cells were transfected with
pEGFP-K85TAG-mCherry and pBhcKRS-4PylT (200 ng each). After
a 20 h incubation at 37 °C/5% CO2 in DMEM with 10% FBS in the
presence of 1 (0.25 mM), cells were washed with DMEM without
phenol red and then incubated for 1 h. Before light activation,
mCherry-expressing cells were identified using the TXRED channel,
and imaged with a Nikon A1Rsi confocal microscope (20× objective,
2-fold zoom, EGFP (ex. 488 nm) and mCherry (ex. 560 nm)
channels). Subsequently, cells were illuminated for 15 s at 365 nm
light (DAPI filter, 358−365 nm), and EGFP and mCherry
fluorescence was acquired by time-lapse imaging (every 1 min for
the first 15 min, every 5 min for the following 150 min, scan resolution
512 × 512, scan zoom 2× , dwell time 1.9 ms). The mean EGFP
fluorescence intensities were quantified using Nikon Elements
software.
ASSOCIATED CONTENT
* Supporting Information
■
S
Protein mass spectrometry, additional micrographs, NMR
spectra, oligonucleotide sequences, and synthesis protocols.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
deiters@pitt.edu.
■
Two-Photon Light Activation of EGFP. HEK 293T cells were
plated into a polylysine-coated μ-dish (ibidi), and after incubation to
50% confluency, the cells were transfected with 1 μg each of pEGFP-
KTAG-mCherry and pBhcKRS-4PylT. After a 20 h incubation at 37
°C/5% CO2 in DMEM with 10% FBS in the presence of 1 or 2 (0.25
mM, 0.5% DMSO), cells were washed with DMEM without phenol
red and then incubated for 1 h. Cells were imaged with an Olympus
Fluoview confocal microscope before two-photon irradiation (40× oil
objective, EGFP (ex. 488 nm) and mCherry (ex. 560 nm) channels),
imaging positions for mCherry-expressing cells were recorded, and the
cell μ-dish was transferred to an Olympus multiphoton microscope for
irradiation (Olympus Fluoview FV1000 MPE). Cells were localized at
the previously recorded positions, focused using the mCherry channel,
and then irradiated using a 760 nm laser (130 mW, 5% of laser power,
30 cycles of scanning, 2 μm/s dwell time, MaiTai DSBB-OL IR pulsed
laser). After irradiation, the cell μ-dish was transferred back to the
original microscope for imaging.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Dustin Lockney for preparation of the pEGFP-
K85TAG plasmid. This work was supported in part by the
National Science Foundation (MCB-1330746, CHE-0848398)
and the University of Pittsburgh. This research used the Center
for Biologic Imaging, the Biomedical Mass Spectrometry
Center and UPCI Cancer Biomarker Facility that are supported
in part by the National Institutes of Health (P30CA047904).
J.W.C. is supported by the Medical Research Council
(U105181009, UD99999908). D.P.N. was supported by a
fellowship from Trinity College.
Mutant PylRS Structure Modeling and Energy Minimization.
The initial template structure of PDB 2Q7H was chosen as a starting
point for all modeling. The missing loops were remodeled using
MODELER and the two point mutations (Y271A and L274M) were
constructed using the mutate_model.py script provided by MODELER.
Superposition of PDB 2Q7G on top of 2Q7H provided the
coordinates for the incorporation of ATP and magnesium ions into
the newly mutated structure. The ATP and magnesium ions were
parametrized in antechamber using previously developed parame-
ters.60,61 The mutated structure was imported into AMBER12 software
using the AMBER FF99SBILDN force field.62 The protein was placed
into a cubic box with a 12.0-Å border, solvated with 17 316 water
molecules, and charge neutralized with the addition of six sodium ions.
This system was energy minimized first with 5000 steps steepest
descent method, followed by 15 000 steps conjugate gradient method
with 5 kcal/mol restraints on all atoms. This was followed by another
5000 steps steepest descent method, followed by 15 000 steps
conjugate gradient method with 2 kcal/mol restraints on all atoms
except Y271A and L274M. The resulting energy-minimized structure
was used as the starting structure for all our docking experiments. All
AMBER12 computational experiments were completed on the Center
for Simulation and Modeling (SAM) Frank supercomputer at the
University of Pittsburgh.
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