A genetically encoded e-N-Methyl lysine in mammalian cells

Full text of DOI:10.1002/cbic.200900690

DOI: 10.1002/cbic.200900690
A Genetically Encoded e-N-Methyl Lysine in Mammalian Cells
Dan Groff,[a] Peng R. Chen,[b] Francis B. Peters,[a] and Peter G. Schultz*[a]
The post-translational methylation of lysine modulates the
activity, stability, localization and biomolecular interactions of
many eukaryotic proteins. For example, monomethylation of
Lys372 in the mammalian tumor suppressor p53 has been
shown to affect protein stability and localization.[1] Protein
methylation plays a particularly important role in gene expres-
sion due to its involvement in the histone code, in which spe-
cific modifications to histone proteins modulate the transcrip-
tional status of specific genes. Methylation of distinct histone
lysine residues has been correlated with both transcriptional
activation and repression depending on the lysine modified.[2]
To better understand the functional consequences of lysine
methylation, methods are needed to generate proteins with
defined methylation status both in vitro and in living cells.
One straightforward in vitro method for lysine methylation
makes use of methyltransferase enzymes,[3] which transfer the
S-methyl group of the cofactor S-adenosyl methionine to the
sidechains of lysine and arginine. These enzymes have very
specific sequence requirements, and their activity can lead to
heterogeneous mixtures of methylation products.[4] To circum-
vent these limitations, one can use solid-phase peptide synthe-
sis to directly incorporate methylated lysine residues into full-
length proteins by using native chemical ligation.[5] In addition,
in vitro translation has been adapted for the production of his-
tone tails with multiple modified lysine residues.[6] However,
these methods can suffer from low yields, restrictions on the
site of modification, and are not easily adapted to cellular stud-
ies. Recently, an in vitro chemical modification strategy was de-
veloped that takes advantage of the unique reactivity of cys-
teine. The reaction between cysteine and N-methyl aminoeth-
ylhalides generates a thioether adduct structurally similar to
methyl lysine. Mono-, di-, and tri-methylated lysine analogues
have been generated in this way (Scheme 1).[7] By using a simi-
lar strategy, unnatural amino acid mutagenesis was used to se-
lectively incorporate phenylselenocysteine into proteins. It can
be subsequently oxidized to dehydroalanine and reacted with
aminoethylthiols to again produce methyl lysine analogues.
However, an initial oxidation step is required, so this strategy is
not compatible with proteins containing redox active cysteine
or methionine residues.[8]
Biosynthetic approaches have also been used to incorporate
methyl lysine into proteins. For example, the e-methyl lysine
precursor Ne-tert-butyl-oxycarbonyl Ne-methyl-l-lysine (Boc
methyl lysine) has been genetically inserted into proteins in
E. coli. This strategy produces good yields of homogeneously
monomethylated proteins largely independent of sequence
context.[4] However, Boc deprotection in aqueous TFA is neces-
sary to generate monomethyl lysine and can cause denatura-
tion and loss of biological activity with many proteins.[9]
All of the above methods for introducing methyl lysine re-
quire in vitro manipulation to produce the final, methylated
protein. This restriction generally prevents studies of methylat-
ed proteins in their native cellular context. To overcome this
limitation, we have genetically encoded the photocaged N-
methyl lysine, Ne-o-nitrobenzyl-oxycarbonyl-Ne-methyl-l-lysine
1, in both bacteria and mammalian cells. Photocaged second
messengers and proteins are widely used tools for the spatial
and temporal control of a variety of cellular processes because
light allows noninvasive generation of the active photoprod-
ucts in the cell. Furthermore, it has been previously demon-
strated that o-nitrobenzyl-O-tyrosine and dimethoxy-o-nitro-
benzyl-O-serine can be efficiently deprotected with light in
E. coli,[10] Xenopus oocytes,[11] and yeast.[12]
The synthesis of photocaged methyl lysine 1 involves two
sequential reductive aminations of Na-tert-butyl-oxycarbonyl-l-
lysine with benzaldehyde and formaldehyde using STABH to
afford Na-tert-butyl-oxycarbonyl-Ne-benzyl-Ne-methyl-l-lysine.[13]
Reductive debenzylation quantitatively yielded Na-tert-butyl-
oxycarbonyl-Ne-methyl-l-lysine 4 which was then coupled to o-
nitrobenzyl chloroformate to produce 5. Boc deprotection pro-
ceeded quantitatively with HCl in dioxane to generate photo-
caged methyl lysine 1.
To genetically encode 1, we used a pyrrolysyl-tRNA synthe-
Pyl
CUA
tase from M. barkeri (MbPylRS) and a pyrrolysyl tRNA (tRNA
)
from M. mazei,[14] which previously were adapted for the site-
specific incorporation of unnatural amino acids into proteins in
response to the amber nonsense codon TAG.[15–17] The ortho-
gonality of this pair to endogenous tRNAs and aminoacyl-tRNA
synthetases (aaRSs) has been demonstrated in both E. coli and
mammalian cells, so that an aaRS evolved to incorporate the
unnatural amino acid Ne-o-nitrobenzyloxycarbonyl-l-lysine in
E. coli can also be used in mammalian cells. An aaRS library
was created in E. coli in which the codons for residues L270,
Y271, L274 and C313 of the pyrrolysyl-tRNA synthetase from
M. barkeri were all randomized as NNK. Directed evolution with
two positive rounds and one negative round of selection as
previously described[18] resulted in the identification of the
aaRS G12 (experimental details in Supporting Information),
which is capable of incorporating 1 into proteins. aaRS G12
has the following mutations: Y271I, L274M, and C313A.
[a] Dr. D. Groff,+ F. B. Peters,+ Prof. Dr. P. G. Schultz
Department of Chemistry, The Scripps Research Institute
10550 N. Torrey Pines, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-9440
E-mail: schultz@scripps.edu
[b] Dr. P. R. Chen+
Department of Chemical Biology, Peking University
Beijing 100871 (China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900690.
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ChemBioChem 2010, 11, 1066 – 1068

Scheme 1. Synthesis of photocaged methyl lysine 1. a) Benzaldehyde and STABH followed by formaldehyde and STABH; b) H2 with Pd/C; c) o-nitrobenzyl chol-
oformate; d) 4n HCl/dioxanes
To determine the fidelity and efficiency with which 1 can be
incorporated into proteins in E. coli, a myoglobin gene with an
the lysine mutant were present (Figure 1A). The methyl lysine
mutant is likely due to enzymatic degradation of the nitroben-
zyl carbamate moiety; this has been observed previously.[15]
The lysine mutant might result from demethylation of methyl
lysine; it was not observed in the mass spectrum in cases in
which protein was expressed in the absence of 1; this demon-
strates that this peak is not due to misincorporation of lysine
amber mutation at codon 99 and a C-terminal His6 tag was ex-
Pyl
CUA
pressed in the presence of the G12 aaRS/tRNA pair and 1. In
the absence of unnatural amino acid, no full length myoglobin
was evident by SDS-PAGE analysis. In Terrific Broth media sup-
plemented with 1 mm 1, the yield of mutant protein was
4.2 mgproteinLꢀ1 (Figure 1A), compared to yields of around
1 mgLꢀ1 for protein expressed with the WT pyrrolysine synthe-
at this position.
Pyl
CUA
Next we determined if the G12 aaRS/tRNA
pair could be
tase and the pyrrolysine analogue Ne-cyclopentyloxycarbonyl-
used to selectively incorporate photocaged methyl lysine into
Tyr
CUA
l-lysine[15] and 2 mgLꢀ1for Myo4TAG suppressed with tRNA
proteins in mammalian cells. The genes encoding G12 and
Pyl
and the WT tyrosyl aminoacyl-tRNA synthetase from M. janna-
schii.[16] To confirm incorporation of 1 at position 99, the
mutant myoglobin was purified with nickel-affinity chromatog-
raphy and then subjected to electrospray mass spectral analy-
sis. For protein expressed in the presence of 1, mass peaks of
18540 Da corresponding to the photocaged methyl lysine
mutant, 18408 Da corresponding to the methyl lysine mutant
with an additional acetylation, and 18351 Da corresponding to
tRNA
were introduced into the mammalian vector pCMV,
CUA
hereafter designated pCMV-G12, with the G12 gene under con-
Pyl
CUA
trol of a constitutive CMV promoter and a single tRNA under
control of a human U6 promoter.[15] Chinese hamster ovary
(CHO) cells were transiently transfected with vector pCMV-G12
and a reporter plasmid containing the eGFP gene with
codon 40 mutated to TAG and a C-terminal His6 tag. CHO cells
grown in media lacking 1 exhibited no visible fluorescence,
whereas cells grown in media with 1 mm 1 produced a bright
GFP signal (Figure 2A). This was further verified with an anti-
His6 western blot (Figure 2B), which indicated that full length
protein was produced only in the presence of photocaged
methyl lysine 1. Finally, to verify that the protein contained
only photocaged methyl lysine, 21 mg of protein was isolated
by nickel affinity chromatography from 2.2ꢁ107 CHO cells.
Electrospray mass spectral analysis showed the presence of
only one peak at 29840 m/z (Figure 2C) corresponding to the
eGFP40TAG!1 mutant (calcd mass=29842 Da). In addition,
the lack of peaks corresponding to N-methyl lysine, lysine, or
additional acetylation further support the hypothesis that
these modifications in E. coli result from enzymes not present
in mammalian cells. To verify that this peak represents photoc-
aged methyl lysine, protein was subjected to photolysis for
20 min with ꢁ 365 nm light, an irradiation regime demonstrat-
ed to be safe for use with mammalian cells.[19] Subsequent
mass spectral analysis showed quantitative conversion to a
protein with a mass of 29663 Da, 177 mass units lighter, corre-
sponding to the loss of one o-nitrobenzyloxycarbonyl group.
To demonstrate that methyl lysine can be liberated in living
cells, eGFP40!1 was photolyzed in CHO cells. The previous
experiment was repeated with irradiation before cell lysis and
protein purification. Two peaks are evident in the mass spec-
trum, a small peak at 29845 m/z corresponding to eGFP con-
Figure 1. Photocaged methyl lysine can be incorporated into proteins with
Pyl
CUA
high fidelity in E. coli using the G12 aaRS/tRNA pair. A) E. coli amber sup-
pression of Myo99TAG in Terrific Broth. Left lane: protein expressed without
1. Right lane: protein expressed with 1 mm 1. B) Electrospray mass spectrum
of TAG99!1 Myo mutant.
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Acknowledgements
This work was supported by NIH grants R01M62159 and
PN22EY01 (P.G.S.).
Keywords: amino acids · methyl lysine · photochemistry ·
post-translational modifications · pyrrolysine · unnatural amino
acids
[1] S. Chuikov, J. K. Kurash, J. R. Wilson, B. Xiao, N. Justin, G. S. Ivanov, K.
McKinney, P. Tempst, C. Prives, S. J. Gamblin, N. A. Barlev, D. Reinberg,
Nature 2004, 432, 353.
[2] P. Hublitz, M. Albert, A. H. Peters, Int. J. Dev. Biol. 2009, 53, 335.
[3] F. Martino, S. Kueng, P. Robinson, M. Tsai-Pflugfelder, F. van Leeuwen, M.
Ziegler, F. Cubizolles, M. M. Cockell, D. Rhodes, S. M. Gasser, Mol. Cell
2009, 33, 323.
[4] D. P. Nguyen, M. M. Alai, P. B. Kapadnis, H. Neumann, J. W. Chin, J. Am.
Chem. Soc. 2009, 131, 14194.
[5] S. He, D. Bauman, J. S. Davis, A. Loyola, K. Nishioka, J. L. Gronlund, D.
Reinberg, F. Meng, N. Kelleher, D. G. McCafferty, Proc. Natl. Acad. Sci.
USA 2003, 100, 12033.
[6] T. J. Kang, S. Yuzawa, H. Suga, Chem. Biol. 2008, 15, 1166.
[7] M. D. Simon, F. Chu, L. R. Racki, C. C. de La Cruz, A. L. Burlingame, B.
Panning, G. J. Narlikar, K. M. Shokat, Cell 2007, 128, 1003.
[8] J. Guo, J. Wang, J. S. Lee, P. G. Schultz, Angew. Chem. Int. Ed. 2008, 47,
6399.
[9] H. Kanazawa, M. Nishikawa, A. Mizutani, C. Sakamoto, Y. Morita-Murase,
Y. Nagata, A. Kikuchi, T. Okano, J. Chromatogr. A 2008, 1191, 157.
[10] A. Deiters, D. Groff, Y. Ryu, J. Xie, P. G. Schultz, Angew. Chem. 2006, 118,
2794; Angew. Chem. Int. Ed. 2006, 45, 2728.
Figure 2. Photocaged methyl lysine 1 can be incorporated into proteins
with high fidelity in mammalian cells. A) CHO expression of eGFP73TAG
transfected with pCMV-G12 imaged with fluorescence microscopy. Top
image shows cells supplemented with 1 mm 1; bottom image shows cells
with no unnatural amino acid. B) Western blot analysis of eGFP40TAG!1
mutant expression probed with anti-His6 antibody. C) Electrospray mass
spectrum of eGFP40TAG!1 mutant protein from CHO cells purified by
using nickel affinity chromotography. Top: before irradiation, middle: after
irradiation ꢁ365 nm for 20 min, bottom: irradiation in CHO cells ꢁ365 nm
for 60 min.
[11] K. D. Philipson, J. P. Gallivan, G. S. Brandt, D. A. Dougherty, H. A. Lester,
Am. J. Physiol. Cell Physiol. 2001, 281, C195.
[12] E. A. Lemke, D. Summerer, B. H. Geierstanger, S. M. Brittain, P. G. Schultz,
Nat. Chem. Biol. 2007, 3, 769.
[13] Z.-P. Huang, X.-Y. Su, J.-T. Du, Y.-F. Zhao, Y.-M. Li, Tetrahedron Lett. 2006,
47, 5997.
[14] G. Srinivasan, C. M. James, J. A. Krzycki, Science 2002, 296, 1459.
[15] P. R. Chen, D. Groff, J. Guo, W. Ou, S. Cellitti, B. H. Geierstanger, P. G.
Schultz, Angew. Chem. 2009, 121, 4112; Angew. Chem. Int. Ed. 2009, 48,
4052.
[16] H. Neumann, S. Y. Peak-Chew, J. W. Chin, Nat. Chem. Biol. 2008, 4, 232.
[17] T. Mukai, T. Kobayashi, N. Hino, T. Yanagisawa, K. Sakamoto, S. Yoko-
yama, Biochem. Biophys. Res. Commun. 2008, 371, 818.
[18] L. Wang, A. Brock, B. Herberich, P. G. Schultz, Science 2001, 292, 498.
[19] W. F. Edwards, D. D. Young, A. Deiters, ACS Chem. Biol. 2009, 4, 441.
taining 1, and a major peak at 29662 m/z corresponding to
photolyzed eGFP containing methyl lysine at position 40.
In conclusion, we have shown that N-methyl lysine can be
efficiently incorporated into proteins in both E. coli and mam-
malian cells with high fidelity. Although additional effort is nec-
essary to produce homogenously methylated proteins in
E. coli, this system is capable of introducing photocaged
methyl lysine site specifically into proteins in mammalian cells.
In addition, we demonstrated that the caging group can be re-
moved in living cells to produce homogeneously methylated
proteins. This method will be a useful tool in understanding
the role of monomethylation in a variety of biological process-
es.
Received: November 12, 2009
Published online on April 26, 2010
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