A genetically encoded aldehyde for rapid protein labelling

Article abstract of DOI:10.1039/c4cc02000f

Using a mutant pyrrolysyl-tRNA synthetase-tRNAPylCUA pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02000f

Showcasing research from the laboratory of Wenshe Liu and
co-workers, Department of Chemistry, Texas A&M University,
Texas, USA
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A genetically encoded aldehyde for rapid protein labelling
A mutant pyrrolysyl-tRNA synthetase-tRNAPCyUl A pair has been used
to incorporate an aldehyde residue into proteins, which could be
rapidly labelled with hydroxylamine-based dyes in vitro (left) and
in vivo (right) at neutral pH. Cover designed by Alfred Tuley.
See Wenshe R. Liu et al.,
Chem. Commun., 2014, 50, 7424.
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A genetically encoded aldehyde for rapid protein
labelling†
Cite this: Chem. Commun., 2014,
50, 7424
Alfred Tuley,a Yan-Jiun Lee,a Bo Wu,a Zhiyong U. Wangb and Wenshe R. Liu*a
Received 17th March 2014,
Accepted 4th April 2014
DOI: 10.1039/c4cc02000f
www.rsc.org/chemcomm
Pyl
CUA
Using a mutant pyrrolysyl-tRNA synthetase-tRNA
pair, 3-formyl-
phenylalanine is genetically incorporated into proteins at amber
mutation sites in Escherichia coli. This non-canonical amino acid
readily reacts with hydroxylamine dyes, leading to rapid and site-
selective protein labelling.
Although the aldehyde is one of the most versatile chemical
functionalities and participates in a variety of useful chemical
reactions,1 it is not a part of the 20 canonical amino acids. To
harness the unique reactivity of aldehydes with hydrazine and
hydroxylamine dyes for protein labelling, aldehyde tags have been
introduced into proteins by oxidizing an N-terminal serine or via
enzymatic modifications of preinstalled substrate peptides.2 The
N-terminal oxidation approach can only be applied in vitro and
the enzymatic approach is generally confined for the aldehyde
installation at the two protein termini. To genetically encode the
keto functionality at any chosen site in a protein, several ketone-
containing non-canonical amino acids (NCAAs) have been designed
and incorporated into proteins in living cells using the amber
suppression approach.3 However, a method for the genetic incor-
poration of an aldehyde-containing NCAA has not been developed.
We are interested in a genetically encoded, aldehyde-containing
NCAA due to its superior reactivity with respect to ketone-
containing NCAAs, and the observation that aniline can selectively
enhance the reaction kinetics of the aldehyde–hydroxylamine/
hydrazine condensation.4
Fig. 1 (A) Structures of 1–4. (B) The synthetic route of 1.
this mutant enzyme to accept diverse meta-substituted phenyl-
alanine substrates, we anticipated that this same mutant enzyme
would also accept 1 as its substrate. To test this prospect, 1 was
readily prepared in a high yield by a three-step synthesis (Fig. 1B).
Its acceptance as a substrate of PylRS(N346A/C348A) was then
examined. An E. coli BL21(DE3) cell that harboured two plasmids,
pEVOL-pylT-PylRS(N346A/C348A) and pET-pylT-sfGFP2TAG, was
employed for the investigation. Plasmid pEVOL-pylT-PylRS(N346A/
C348A) contains genes coding both PylRS(N346A/C348A) and
tRNAPCyUlA. Plasmid pET-pylT-sfGFP2TAG carries a tRNA
coding
Pyl
CUA
gene and a sequence-optimized superfolder green fluorescent
protein (sfGFP) gene with an amber mutation at its S2 position.
Growing cells in M9 minimal medium supplemented with 1%
glycerol, 1 mM IPTG and 0.2% arabinose, but without 1, afforded
a minimal expression level of full-length sfGFP (o1 mg Lꢀ1).
Providing 1 at 2 mM into the medium promoted full-length
sfGFP expression (Fig. 2A). The expression level is comparable to that
of para-propargyloxy-phenylalanine (ProY), and the electrospray ioni-
zation mass spectrometry analysis of the purified 1-containing sfGFP
displayed a molecular weight that agreed well with its theoretical
value (Fig. 1B). Moreover, compound 1 is not toxic to E. coli cells, as
phenotypes and cell pellets are comparable to control experiments.
Dawson et al. previously showed that aniline could serve as an
excellent catalyst for speeding the imine formation of aldehydes with
hydroxylamines and hydrazines. To demonstrate this catalytically
In our previous studies, we revealed that a mutant pyrrolysyl-tRNA
synthetase with mutations N346A/C348A (PylRS(N346A/C348A))
recognizes a number of phenylalanine derivatives including meta-
Pyl
acetyl-phenylalanine (2 in Fig. 1A) and, in coordination with tRNA
,
CUA
directs their genetic incorporation at amber mutation sites in
Escherichia coli and mammalian cells.5 Given the size similarity
between 2 and meta-formyl-phenylalanine (1), and the ability of
a Department of Chemistry, Texas A&M University, College Station, TX 77843, USA.
E-mail: wliu@chem.tamu.edu
b Department of Chemistry and Physics, Troy University, Troy, Alabama, 36081, USA
† Electronic supplementary information (ESI) available: Synthesis, protein expres-
sion, protein labelling, and mass spectrometry analysis. See DOI: 10.1039/c4cc02000f rapid reaction on proteins, sfGFP with 1 incorporated at its N149
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Fig. 2 (A) The site-specific incorporation of 1 into sfGFP at its S2 position. Cells
transformed with pEVOL-PylRS(N346A/C348A) and pET-pylT-sfGFP2TAG
were grown under conditions with or without a NCAA supplement. (B) The
deconvoluted ESI-MS spectrum of the purified 1-containing sfGFP. Its
theoretical value is 27756 Da.
¨
position (sfGFP-1) was expressed and then applied to a Forster
resonance energy transfer (FRET)-based kinetic investigation of
its reaction with a coumarin dye 3,3d,6 in the presence of 100 mM
aniline and at pH 7 using a fluorescence spectrophotometer
(Fig. 1A). 3 has an excitation wavelength at 417 nm and emits at
476 nm. Its emitted light falls in the range of excitation lights of
sfGFP that emits at 510 nm.7 The reaction of sfGFP-1 with 3 will
place 3 in a close locality of the sfGFP fluorophore for FRET.
All kinetic analyses were carried out under pseudo-first-order
conditions in which 3 was at least in 5-fold excess in comparison
Fig. 3 (A) Fluorescent intensities as a function of time for reactions of 0.05 mM
sfGFP-1 (coloured in blue) and 0.05 mM sfGFP-2 (coloured in red) with 10 mM 3
in the presence of 100 mM aniline and at pH 7. Presented in the inset is the
linear dependence of the determined pseudo first-order rate constants for the
reaction of 0.1 mM sfGFP-1 with 3 on the concentrations of 3. (B) Gel imaging
analysis of labelling of sfGFP-1 and sfGFP-2 with 4 with different incubation
times. For both sfGFP-1 and sfGFP-2, labelling was carried out under condi-
tions with 8 mM protein, 10 mM 4, 100 mM aniline, and pH 7. Reactions were
quenched with the addition of 2 mM benzaldehyde at indicated times and
analyzed by denaturing SDS-PAGE. The top panel shows proteins with
Coomassie blue staining and the bottom panel presents the fluorescent
imaging of the same gel before Coomassie blue staining.
to sfGFP-1. Data collected under these conditions were well fitted
0
to a single exponential increase equation F = F1 ꢀ F2 ꢁ e(ꢀk ꢁt)
,
where F was the detected fluorescent signal at a given time, F1
was the final fluorescence, F1 ꢀ F2 was the background fluor-
escent signal, and k0 was the apparent pseudo first-order rate
constant (Fig. 3A). The determined k0 values were plotted against
the concentrations of 3 and fitted to the equation k0 = k ꢁ [3] + C,
where k was a second-order rate constant for the reaction of
sfGFP-1 with 3 (inset of Fig. 3A). The calculated second-order rate Reactions were quenched at different times and analyzed by
constant was 182 ꢂ 12 Mꢀ1 sꢀ1. This determined rate constant is denaturing SDS-PAGE to remove the intrinsic fluorescence of
comparable to what Dawson et al. reported for aniline-catalyzed sfGFP. Fluorescent imaging of two proteins at different labelling
bioconjugation, and is among one of the most rapid bioorthogonal times clearly showed that sfGFP-1 was efficiently labelled at 25 min
reactions.4,8 We also carried out the kinetic analysis in the absence but sfGFP-2 was minimally labelled even at 2 h. Labelling could
of aniline. However, no obvious signal changes were observed also be achieved in the absence of aniline, though reaction times
when 0.1 mM sfGFP-1 reacted with 4 mM 3 in a period of 2 h, were significantly longer (Fig. S2, ESI†). Once again, sfGFP-1
indicating very slow kinetics of the sfGFP-1 reaction with 3 in the showed superior labelling with respect to sfGFP-2. This result
absence of aniline and at pH 7. We also tried much higher shows that, in systems where aniline may be toxic, labelling can
concentrations of 3 to speed up the reaction rate. However, the still be achieved without it or at much lower concentrations.
background fluorescence from 3 under these conditions was too
With the demonstration of fast labelling kinetics of 1 with
high to collect reliable data. As a comparison, a sfGFP variant with hydroxylamine dyes, we then proceeded to test the conditions
2 incorporated at its N149 position (sfGFP-2) was also expressed for labelling proteins that bear site-specifically incorporated 1
and used for kinetic investigation of its reaction with 3 in the in living cells. Plasmid pEVOL-pylT-PylRS(N346A/C348A) and a
presence of 100 mM aniline and at pH 7. As shown in Fig. 3A, previously constructed plasmid, pETDuet-OmpXTAG, were
under the tested conditions of 0.05 mM sfGFP-2 and 10 mM 3, no used to transform E. coli BL21 cells. The latter plasmid con-
obvious signal increase due to the sfGFP-2 reaction with 3 was tained a gene coding for an E. coli outer membrane protein
observed in a period of 30 min. The fluorescence decrease was OmpX,8b with an AAAXAA (A denotes alanine and X denotes an
caused by the bleaching effect of the excitation light.
amber mutation) insertion between two extracellular residues,
The different labelling kinetics of sfGFP-1 and sfGFP-2 were also 53 and 54. The transformed cells were grown in M9 minimal
examined by fluorescent imaging of SDS-PAGE analyzed 4-labelled medium supplemented with 1% glycerol, 1 mM IPTG, 0.2%
products. Both sfGFP-1 and sfGFP-2 at 8 mM were incubated arabinose, and 2 mM 1 or 2 to express OmpX with 1 or 2
with 10 mM 4 in the presence of 100 mM aniline and at pH 7. incorporated (OmpX-1 or OmpX-2). Cells were then harvested
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We are thankful for the financial support from the National
Institute of Health (grant 1R01CA161158), the National Science
Foundation (grant CHE-1148684), and the Welch Foundation
(grant A-1715). We also thank Dr Yohannes H. Rezenom from
the Laboratory for Biological Mass Spectrometry at Texas A&M
University for characterizing our proteins with electrospray
ionization mass spectrometry.
7426 | Chem. Commun., 2014, 50, 7424--7426
This journal is ©The Royal Society of Chemistry 2014