Unexpected preference of the E. coli translation system for the ester bond during incorporation of backbone-elongated substrates

Article abstract of DOI:10.1021/ja068033n

There have been recent advances in the ribosomal synthesis of various molecules composed of nonnatural ribosomal substrates. However, the ribosome has strict limitations on substrates with elongated backbones. Here, we show an unexpected loophole in the E. coli translation system, based on a remarkable disparity in its selectivity for β-amino/hydroxy acids. We challenged β-hydroxypropionic acid (β-HPA), which is less nucleophilic than β-amino acids but free from protonation, to produce a new repertoire of ribosome-compatible but main-chain-elongated substrates. PAGE analysis and mass-coupled S-tag assays of amber suppression experiments using yeast suppressor tRNA^Phe_CUA confirmed the actual incorporation of β-HPA into proteins/oligopeptides. We investigated the side-chain effects of β-HPA and found that the side chain at position α and R stereochemistry of the β-substrate is preferred and even notably enhances the efficiency of incorporation as compared to the parent substrate. These results indicate that the E. coli translation machinery can utilize main-chain-elongated substrates if the pK_a of the substrate is appropriately chosen.

Full text of DOI:10.1021/ja068033n

Published on Web 04/20/2007
Unexpected Preference of the E. coli Translation System for
the Ester Bond during Incorporation of Backbone-Elongated
Substrates
Shinsuke Sando,* Kenji Abe, Nobuhiko Sato, Toshihiro Shibata,
Keigo Mizusawa, and Yasuhiro Aoyama*
Contribution from the Department of Synthetic Chemistry and Biological Chemistry, Graduate
School of Engineering, Kyoto UniVersity, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
Received November 9, 2006; E-mail: ssando@sbchem.kyoto-u.ac.jp; aoyamay@sbchem.kyoto-u.ac.jp
Abstract: There have been recent advances in the ribosomal synthesis of various molecules composed
of nonnatural ribosomal substrates. However, the ribosome has strict limitations on substrates with elongated
backbones. Here, we show an unexpected loophole in the E. coli translation system, based on a remarkable
disparity in its selectivity for â-amino/hydroxy acids. We challenged â-hydroxypropionic acid (â-HPA), which
is less nucleophilic than â-amino acids but free from protonation, to produce a new repertoire of ribosome-
compatible but main-chain-elongated substrates. PAGE analysis and mass-coupled S-tag assays of amber
suppression experiments using yeast suppressor tRNAPheCUA confirmed the actual incorporation of â-HPA
into proteins/oligopeptides. We investigated the side-chain effects of â-HPA and found that the side chain
at position R and R stereochemistry of the â-substrate is preferred and even notably enhances the efficiency
of incorporation as compared to the parent substrate. These results indicate that the E. coli translation
machinery can utilize main-chain-elongated substrates if the pKa of the substrate is appropriately chosen.
biopolymers.18-20 This is expected to allow the selection and
evolution of nonnatural molecules in combination with ribosome-
Introduction
The ribosome is a highly sophisticated RNA machine that
directs the mRNA-templated process of amino acid polymeri-
zation, which ultimately produces size-, shape-, and sequence-
defined polymers (i.e., proteins). Although the ribosomal system
has evolved for the synthesis of proteins, extensive research
has revealed a wide substrate acceptance beyond the 20
canonical amino acids.1-4 Much recent effort has focused on
the use of the ribosomal translation system for the synthesis
of sequence-programmed peptidomimetics5-17 or nonnatural
display21-23 or mRNA-display24,25 technologies.
Extensive work has indicated the remarkable tolerance of the
translation system for side-chain modifications of its substrates
(amino acids).2 Particularly intriguing in this context are the
permissible main-chain or backbone modifications, which should
undoubtedly extend the structural diversity of decodable librar-
ies. A recent study using a reconstituted translation system has
revealed the amino acid backbone specificity of the Escherichia
coli translation machinery.8 It clearly confirmed that various
types of R-amino acid analogues, including N-methyl amino
acids or R-hydroxy acids, can be incorporated into oligopeptides
by the ribosome. However, homologous â-amino acids were
found to be much less efficient substrates as compared to their
R counterparts.8,16 The incorporation of the former seems to be
governed by the surrounding codons, tRNA sequences, or even
(1) Wang, L.; Schultz, P. G. Angew. Chem., Int. Ed. 2004, 44, 34-66.
(2) England, P. M. Biochemistry 2004, 43, 11623-11629.
(3) Strømgaard, A.; Jensen, A. A.; Strømgaard, K. ChemBioChem 2004, 5,
909-916.
(4) Hendrickson, T. L.; de Cre´cy-Lagard, V.; Schimmel, P. Annu. ReV. Biochem.
2004, 73, 147-176.
(5) Forster, A. C.; Weissbach, H.; Blacklow, S. C. Anal. Biochem. 2001, 297,
60-70.
(6) Forster, A. C.; Tan, Z.; Nalam, M. N. L.; Lin, H.; Qu, H.; Cornish, V. W.;
Blacklow, S. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6353-6357.
(7) Forster, A. C.; Cornish, V. W.; Blacklow, S. C. Anal. Biochem. 2004, 333,
358-364.
(16) Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H. Nat. Methods 2006, 3, 357-
359.
(8) Tan, Z.; Forster, A. C.; Blacklow, S. C.; Cornish, V. W. J. Am. Chem.
Soc. 2004, 126, 12752-12753.
(17) Sando, S.; Kanatani, K.; Sato, N.; Matsumoto, H.; Hohsaka, T.; Aoyama,
Y. J. Am. Chem. Soc. 2005, 127, 7998-7999.
(18) Link, A. J.; Mock, M. L.; Tirrell, D. A. Curr. Opin. Biotechnol. 2003, 14,
603-609.
(9) Tan, Z.; Blacklow, S. C.; Cornish, V. W.; Forster, A. C. Methods 2005,
36, 279-290.
(10) Li, S.; Millward, S.; Roberts, R. W. J. Am. Chem. Soc. 2002, 124, 9972-
(19) Kwon, I.; Kirshenbaum, K.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125,
7512-7513.
9973.
(11) Frankel, A.; Li, S.; Starck, S. R.; Roberts, R. W. Curr. Opin. Struct. Biol.
2003, 13, 506-512.
(20) Link, A. J.; Tirrell, D. A. Methods 2005, 36, 291-298.
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4942.
(12) Frankel, A.; Millward, S. W.; Roberts, R. W. Chem. Biol. 2003, 10, 1043-
1050.
(22) Amstutz, P.; Forrer, P.; Zahnd, C.; Plu¨ckthun, A. Curr. Opin. Biotechnol.
2001, 12, 400-405.
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28, 159-165.
(23) He, M.; Taussig, M. J. Nucleic Acids Res. 1997, 25, 5132-5134.
(24) Roberts, R. W.; Szostak, J. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
12297-12302.
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127, 11727-11735.
(15) Hartman, M. C.; Josephson, K.; Szostak, J. W. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 4356-4361.
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1997, 414, 405-408.
9
6180
J. AM. CHEM. SOC. 2007, 129, 6180-6186
10.1021/ja068033n CCC: $37.00 © 2007 American Chemical Society

Unexpected Preference of the E. coli Translation System
A R T I C L E S
conditions of translation, indicating severe limitations on main-
chain-elongated substrates. Although modification of the ribo-
some26 or a postsynthetic conversion strategy27 has allowed the
production of nonnatural peptides with additional backbone
residues, the only examples of incorporated â-amino acid
derivatives are hydrazinophenylalanine28 and aminooxyacetic
acid.29 Peptide homologues with one extra carbon, such as
â-peptides, â-esters, or a mixture of these, are found in many
natural products or pharmaceutical agents and could act as the
building blocks for unique structural motifs.30,31 Therefore, a
continuing challenge in the production of a structurally diverse
library is to find a rational strategy for extending the acceptable
backbone modifications, with a variety of side chains.
During our systematic progress toward this goal, we have
identified an unexpected loophole in the E. coli translation
system. Here, we report that a pKa-based approach, which was
initially designed based on the hypothesized mechanism of the
peptidyl transfer (PT) reaction (vide infra), allows the addition
of nonnatural substrates with elongated backbones to the
repertoire of the E. coli translation/polymerization system.
Surprisingly, â-hydroxypropionic acid (â-HPA), which is less
nucleophilic than â-amino acids but free from protonation, is
compatible with the E. coli translation machinery, suggesting
that the E. coli translation system is more suitable for ester-
bond formation than for natural-type amide-bond formation
during the incorporation of nonnatural substrates with elongated
backbones. We also systematically investigated the effects of
the side-chain position on the â-substrate and identified for the
first time the preferred position, which allows the diversification
of substrates even with higher incorporation efficiency. These
new findings could overcome the limitations of backbone
modifications and give a new direction to the rational design
of acceptable substrates with elongated main chains.
Figure 1. Glycine and nonnatural substrates used in this study.
for glycine ethyl ester and â-alanine ethyl ester, respectively33).
We hypothesized that the less efficient incorporation of â-amino
acids is caused by inefficient deprotonation, possibly because
of the nonadjustable positioning or incompatible pKa of the
â-amino group at the A-site. If this hypothesis is correct,
hydroxyalkanoic acid could act as an expanded substrate for
the ribosome, even with an elongated backbone chain, because
the hydroxy group, which occurs in the -OH form under
physiological conditions,34 could attack the carbonyl carbon on
the P-site peptidyl-tRNA without the deprotonation step.
â-Hydroxypropionic acid is not expected to be a good substrate
because of its lower nucleophilicity as compared to the â-amino
acids. However, initially based on this design principle, we
focused on hydroxyalkanoic acids as putative candidates.
Adaptability of Amino Acid Derivatives with Elongated
Backbones. Substrate adaptability to the E. coli translation
machinery was investigated using the nonsense suppression
method.35,36 Amino acids with hydrophobic side chains tend to
be incorporated with much higher efficiency. Therefore, to avoid
any unrevealed side-chain effects of the backbone-elongated
substrates, we initially used simple (side-chain-free) glycine
(Gly) and its analogues/homologues (Figure 1). The substrate
set in this initial study included analogous R-hydroxyacetic acid
(R-HAA) and homologous â-alanine (â-Ala) and â-hydrox-
ypropionic acid (â-HPA), which was prepared by a ring-opening
reaction of â-propiolactone. As representatives of efficiently
acceptable nonnatural substrates (positive controls), R-hydroxy-
3-phenylpropionic acid (R-HPPA, 3-phenyllactic acid) and
Results
Initial pKa-Based Substrate Design. The reason for the
inefficient incorporation of â-amino acids is still unclear.
However, judging from the incorporation of aminooxyacetic acid
into proteins,29 which was confirmed by the selective cleavage
of the N-O bond with zinc dust, the steric hindrance of
elongated main chains seems not to be a critical or determining
factor in the failure of incorporation. One possible explanation
is an incompatibility with the ribosome-catalyzed PT mecha-
nism. The pKa value of the R-amino group of the aminoacylated
tRNA at the A-site, which acts as a nucleophile, is thought to
be approximately 8.1 (25 °C),32 so that deprotonation of the
R-amino group from the ammonium to amino form must be
essential in promoting the PT reaction during the multistep
process of the peptide-bond-forming reaction. This initial
deprotonation step might be more critical for â-amino acids
because the pKa of the â-amino group is typically higher than
that of its R counterpart (pKa ) 7.73 (25 °C) and 9.13 (25 °C)
2-naphthylalanine (Nap) were chosen. These substrates were
chemically misacylated on suppressor yeast tRNAPhe
.
37
CUA
We first checked the hydrolytic stability of the model acyl
esters under translation conditions (50 mM potassium phosphate,
2.5 mM MgCl2, 6.5 mM Mg(OAc)2, 5 mM ammonium acetate,
pH 7.3). HPLC analysis showed that glycinyl-pdCpA under-
went deacylation at 37 °C with a rate constant of 3.5 ( 0.2 ×
10-2 min-1 (half-life, ∼20 min). The hydrolysis rates for the
(33) Edsall, J. T.; Blanchard, M. H. J. Am. Chem. Soc. 1933, 55, 2337-2353.
(26) Dedkova, L. M.; Fahmi, N. E.; Golovine, S. Y.; Hecht, S. M. J. Am. Chem.
Soc. 2003, 125, 6616-6617.
(34) The pKa of aliphatic alcohols is g15, and that of their conjugate acids is
+
e0 (e.g., those for CH3CH2OH and CH3CH2OH2 are 15.9 and -2.4,
(27) Seebeck, F. P.; Szostak, J. W. J. Am. Chem. Soc. 2006, 128, 7150-7151.
(28) Killian, J. A.; van Cleve, M. D.; Shayo, Y. F.; Hecht, S. M. J. Am. Chem.
Soc. 1998, 120, 3032-3042.
respectively (Vollhardt, K.P.C.; Schore, N.E.W.H. Organic Chemistry;
Freeman and Co.: New York, 1994; Chapter 8)). Therefore, there is no
doubt that the substrate (hydroxy acid) exists in the neutral OH state.
(35) Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science
1989, 244, 182-188.
(29) Eisenhauer, B. M.; Hecht, S. M. Biochemistry 2002, 41, 11472-11478.
(30) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001, 101,
3219-3232.
(31) Albertsson, A. C.; Varma, I. K. Biomacromolecules 2003, 4, 1466-1486.
(32) Sievers, A.; Beringer, M.; Rodnina, M. V.; Wolfenden, R. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 7897-7901.
(36) Bain, J. D.; Diala, E. S.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.
Am. Chem. Soc. 1989, 111, 8013-8014.
(37) Heckler, T. G.; Chang, L. H.; Zama, Y.; Naka, T.; Chorghade, M. S.; Hecht,
S. M. Biochemistry 1984, 23, 1468-1473.
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A R T I C L E S
Sando et al.
carbon-elongated main chain is a substrate of the ribosomal
translation system.
S-Tag-Based Suppression Assay Coupled to Direct Mass
Analysis. To confirm further the incorporation of â-HPA at the
amber (UAG) codon, we devised a peptide-based suppression
assay system coupled to direct mass analysis. mRNAs encoding
the 31-mer oligopeptide fMDYKDDDDKQKLXLTHKET-
AAAKFERQHMDS (oligopepY when X is Y (tyrosine) and
oligopepamber when X is a natural/nonnatural substrate assigned
to the UAG amber codon at position 13) were prepared for this
assay. The N-terminal 8-mer (underlined) and the C-terminal
16-mer regions are identical to the FLAG-tag and S-tag
sequences, respectively, and these two tags were placed before
or after the digested E. coli DHFR sequence around position
111 (from position 108 to 114) (italicized). The S-tag peptide
forms a complex with S-protein to restore ribonuclease S
activity. The S-tag sequence was positioned on the C-terminal
side of the UAG amber codon (oligopepamber mRNA) so that
the read-through efficiency could be estimated from the ribo-
nuclease S activity recovered. Ribonuclease S activity was
calculated by measuring the fluorescence increase derived from
a digested fluorescent resonance energy transfer (FRET) oli-
goribonucleotide probe, and the yield of full-length oligopeptide
was determined by comparing the fluorescence increase with
that of various dilutions of oligopeptide translated from oli-
gopepY mRNA (calibration set), as shown in Figure S2 in the
Supporting Information. The S-tag-assay-based ranking of
acceptable nonnatural substrates is consistent with that obtained
by the gel-based assay of full-length DHFR. â-HPA produced
a full-length oligopeptide with a yield of 32% ( 8% relative to
that of the peptide translated from oligopepY mRNA (nonacy-
lated tRNAPheCUA, the negative control, produced ∼6% of full-
length oligopeptide).
Figure 2. Incorporation of amino/hydroxy acids into position 111 of E.
coli DHFR. (A) Western blot analysis of the production of full-length DHFR
in the presence of suppressor tRNAPhe
misacylated with the amino/
CUA
hydroxy acids indicated in the figure. (B) Relative yields of full-length
DHFR. “Non” (or “nonacylated”) indicates the presence of suppressor tRNA
produced by the ligation of yeast tRNAPheCUA-CA with nonacylated pdCpA.
R-HAA and â-Ala were slower, at 2.6 ( 0.1 × 10-3 min-1
(half-life, ∼265 min) and 3.5 ( 0.2 × 10-3 min-1 (half-life,
∼200 min), respectively. â-HPA was remarkably resistant to
hydrolysis (1.7 ( 0.1 × 10-4 min-1), and its half-life reached
∼4000 min. There is no doubt that the stable â-HPA-tRNA is
suitable for long-term translation. However, in this report, the
translation experiment was performed for 60 min (37 °C), so
that the differences in these stabilities can be practically omitted
from the factors affecting substrate adaptability. The ligation
yields of acylated pdCpAs with yeast tRNAPheCUA-CA were also
estimated by 8% polyacrylamide gel analysis and were almost
the same for these nonnatural substrates (Figure S1 in the
Supporting Information).
The FLAG tag at the N-terminus was used to isolate the
translated peptide products, allowing us to characterize the
incorporated nonnatural amino acids at the UAG codon by
MALDI-TOF mass analysis (Figure 3). The oligopepY mRNA
produced an oligopeptide with a single mass peak at 3786.0
Da ([M + H]+ calcd ) 3785.8) (Figure 3A). Selective
incorporation of â-HPA at the UAG codon of oligopepamber
should produce a resultant nonnatural peptide with a mass of
3694.7 and a mass shift for â-HPA-Tyr of -91 Da. Actually,
the addition of â-HPA-tRNACUA resulted in the appearance
of a major mass peak at 3694.5 (Figure 3B). Alkaline hydrolysis
(∼10% aqueous ammonium hydroxide, 37 °C for 24 h) of the
isolated product yielded two new peaks at 1541.3 and 2171.9,
which are identical to the fragments that result from the
hydrolytic cleavage of the ester bond between residues 12 and
13 (calcd 1541.7 and 2172.1, respectively), thus clearly con-
firming the site-selective incorporation of â-HPA (Figure 3D).
The incorporation of â-HPA was further supported by the
following experiments. A unique chemical property of â-HPA
is the remarkable resistance of the acyl ester to hydrolysis (half-
life 4000 min, vide supra). Long preincubation (2200 min) of
In an initial screening, substrate adaptability was examined
by SDS-PAGE analysis using E. coli dihydrofolate reductase
(DHFR) (Figure 2). Wild-type (dhfrwild) or amber-mutated
(dhframber111 with an amber (UAG) mutation at codon position
111) mRNA for DHFR was translated (37 °C for 60 min) in
the presence or absence of the chemically misacylated yeast
37
tRNAPhe
.
At position 111 of DHFR, hydrophobic Nap or
CUA
R-HPPA could be incorporated with expected good yields of
g90% and 60%, respectively, under our experimental conditions
(0.45 µg/µL suppressor tRNA). The less hydrophobic Gly
showed an efficiency of 45%. The analogous R-HAA was also
incorporated at this amber codon with moderate efficiency (35%
( 6%). However, incorporation of the homologous â-Ala was
borderline at this codon position, and the yield of full-length
DHFR was almost the same as that obtained in the presence of
nonacylated tRNA, used as a reference (typically e5%), in
accordance with a recent report.8 In marked contrast, a distinct
band of full-length DHFR was surprisingly produced from
dhframber111 mRNA in the presence of â-HPA-tRNAPheCUA (21%
( 2%), indicating that the â-hydroxy homologue with a one-
acylated tRNAPhe
at 37 °C in translation buffer decreased
CUA
the suppression yield for the incorporation of Nap (85%f5%,
Figure 4). R-Hydroxypropionic acid (L-lactic acid), a structural
isomer of â-HPA with the same mass value, also resulted in a
decrease in the suppression yield (74%f22%). In marked
contrast, the suppression yield for â-HPA-tRNA was not
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Unexpected Preference of the E. coli Translation System
A R T I C L E S
Figure 5. â-HPA derivatives with methyl side chains.
oligopeptides produced gave a single peak with a mass of
3750.9, which is not consistent with an oligopeptide containing
â-Ala but corresponds to the incorporation of Gln (Q) at the
UAG codon, probably by natural tRNAGln ([M + H]+ calcd )
3750.7) (Figure 3C).38 This confirms that simple â-amino acids
are not accepted at this codon, at least under these experimental
conditions.
Side-Chain Effect of Nonnatural Substrates with Elon-
gated Backbones. With a ribosome-compatible main-chain-
elongated substrate (â-HPA) in hand, we moved on to inves-
tigate the side-chain effect on the â-substrate. A permissive side-
chain modification could further enrich the diversity of decodable
libraries and might also improve the incorporation efficiency,
as was revealed for the R-amino acids. However, information
on this phenomenon is still unclear.8,28,29,39 To this end, four
â-HPA derivatives with a -CH3 (methyl) side chain at the R
or â position, with R or S stereochemistry, were prepared and
used in this study (Figure 5). These substrates were chemically
misacylated on yeast tRNAPheCUA. After confirming that the
ligation yields of these substrates are almost identical (Figure
S1 in the Supporting Information), we subjected them to
S-tagged-peptide-based suppression experiments using the oli-
gopepamber template (Figure 6A). Typically, a side chain at â-(S)
has been considered the preferred position. Interestingly,
however, our results indicate that R-(R) is the position most
compatible with the E. coli translation system. A side chain at
R-(R) enhanced the suppression efficiency to 64% ( 8%, which
is almost double that of â-HPA with no side chain (32%).
FLAG-tag-based fishing and MALDI mass analysis also sup-
ported the sequence-selective incorporation of R-(R)-methyl-
â-HPA (calcd 3708.7, found 3708.9). tRNAPheCUA acylated with
R-(R)-methyl-â-HPA maintained this suppression efficiency,
even after the preincubation treatment (37 °C, 2200 min) in
translation buffer (57%f56%, Figure 4), further confirming the
incorporation of the R-(R)-methyl-â-HPA substrate. In addition
to the R configuration, a side chain at R-(S) with opposite
stereochemistry also allowed moderate incorporation (32% (
8%, Figure 6A), although the incorporation efficiency was not
enhanced relative to that of simple â-HPA. To check the
retention of chirality during the synthetic scheme, we prepared
R-(R or S)-methyl-â-HPA-puromycin aminonucleoside (PANS)
by dicyclohexylcarbodiimide (DCC) coupling of synthesized
O-DMTr-protected R-(R or S)-methyl-â-HPA with PANS,
followed by the deprotection of the DMTr group with ∼10%
trifluoroacetic acid in CHCl3 and rough HPLC purification. The
chirality of the R-methyl substituent on the â-HPA substrate
was then assessed by nuclear magnetic resonance (NMR) using
PANS as the internal chiral shift reagent. The protons on
R-methyl-â-HPA scaffold of R-(S)-methyl-â-HPA-PANS are
distinguishable from those of R-(R)-methyl-â-HPA-PANS
(Figure S3 in the Supporting Information), and the
Figure 3. MALDI-TOF mass analysis of oligopeptides translated from
(A) oligopepY and oligopepamber mRNA in the presence of suppressor tRNA
chemically misacylated with (B) â-HPA or (C) â-Ala. (D) shows MALDI
mass peaks obtained from sample (B) after alkaline treatment (∼10%
ammonium hydroxide, 37 °C for 24 h).
Figure 4. Preincubation effect of suppressor tRNA acylated with various
substrates. After the ligation reaction, full-length suppressor tRNAPhe
CUA
misacylated with the substrates indicated in the figure was dissolved (2.5
µg/µL) in translation buffer (50 mM potassium phosphate, 2.5 mM MgCl2,
6.5 mM Mg(OAc)2, 5 mM ammonium acetate, pH 7.3), incubated at 37 °C
for 0 or 2200 min, and directly subjected to a typical translation reaction
(4 µg/10.6 µL) using oligopepamber mRNA. The relative yields of full-length
oligopeptide were determined by an S-tag-based assay.
affected by the long preincubation of 2200 min (38%f42%).
These results indicate the actual incorporation of â-HPA and
preclude the possibility of misincorporation of the structural
isomer. â-Ala-tRNA generated the full-length peptide with a
yield of e5%, which is almost equal to that achieved with
nonacylated suppressor tRNA (∼6%). Mass analysis of the
(38) Nilsson, M.; Ryde´n-Aulin, M. Biochim. Biophys. Acta 2003, 1627, 1-6.
(39) Starck, S. R.; Qi, X.; Olsen, B. N.; Roberts, R. W. J. Am. Chem. Soc.
2003, 125, 8090-8091.
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A R T I C L E S
Sando et al.
incorporated into E. coli DHFR (Figure 6B). Moreover, the
incorporation yields for R-(R)-methyl-â-HPA were higher than
those for simple â-HPA at all of the sites tested, although the
actual increase in the yield depended on the position of the
amber codon.
Discussion
The data described above produced two major previously
unknown and unexpected findings. The first concerns the
versatility of the ribosomal translation system for substrates with
elongated backbones. A gel-based assay and a newly designed
S-tag-based suppression assay system, which is directly coupled
to mass analysis, led us to conclude that â-HPA and its
derivatives constitute a new repertoire for the E. coli translation
system. We confirmed that the ribosome can accept backbone-
extended (by one carbon) substrates and that the active pocket
of the ribosomal PT center is sterically wide enough to accept
main-chain-elongated substrates, supporting earlier findings and
recent experiments.28,29,39 Interestingly, the ribosome shows
remarkable contrasts in its selectivity for R-amino/hydroxy acids
and â-amino/hydroxy acids. Both R-amino acids and R-hydroxy
acids can be substrates of the E. coli translation machinery with
almost the same incorporation efficiencies (Figure 2). In marked
contrast, the E. coli translation system preferentially incorporates
â-hydroxy acids over â-amino acid, as confirmed by a mass-
analysis-coupled S-tag assay (Figure 3). Other examples of
successfully incorporated â-substrates are aminooxyacetic acid29
with pKa ) 4.67 at 23 ( 2 °C41 and hydazinophenylalanine28
(pKa ) 5.97 at 25 °C for R-hydrazinoacetic acid ethyl ester42).
They are also along the pKa-based design strategy. The
acceptable â-substrates including â-HPA share a common
feature, that is, low basicity with pKa < 7. We thus suggest
that the E. coli translation system can incorporate main-chain
elongated substrates when the pKa of the nucleophilic group is
appropriate.
The second finding concerns the compatibility of the E. coli
translation system with side-chain modifications on the â-sub-
strate. The side chain at â-(S) (position â and stereochemistry
S), which has the same position and stereochemistry of the
nucleophilic group and side chain as the natural-type R-amino
acids, has been considered to be the preferred position. However,
our results indicate that the E. coli translation machinery has
an unexpected side-chain preference for the R position and R
stereochemistry at this codon. This appropriate side chain not
only permits incorporation but also enhances the efficiency of
incorporation of the substrate, similar to that of the R-(S) side
chain of the natural R-amino acids. The incorporation efficiency
of R-(R)-methyl-â-HPA was enhanced at all of the sites tested
as compared to that of simple â-HPA (Figure 6B). Interestingly,
at position 10 of E. coli DHFR, the incorporation efficiency
was even better than that of Nap, indicating that R-(R)-
substituted â-HPA is an excellent substrate at this position. It
should also be noted that R-(S)-hydrazinophenylalanine, previ-
ously reported by Hecht et al., has a side chain at this most
preferred position.28 Because the ribosome-catalyzed PT reaction
is thought to be mainly entropy driven,43 it is reasonable to think
Figure 6. (A) Relative yields of full-length oligopeptides translated from
oligopepamber mRNA in the presence of suppressor tRNAPheCUA misacylated
with â-HPA or its derivatives indicated in the figure. The yields were
determined by an S-tag-based assay. Lower panel shows the MALDI mass
data for oligopeptides translated in the presence of suppressor tRNAPhe
CUA
misacylated with R-(R). (B) Western blot analysis of the production of full-
length E. coli DHFR from dhframber10,111,or140 mRNA templates in the
presence of suppressor tRNAPheCUA misacylated with â-HPA, R-(R), â-Ala,
or Nap. (-), the absence of suppressor tRNA; “non” (or “nonacylated”),
the presence of suppressor tRNA produced by the ligation of yeast
tRNAPheCUA-CA with nonacylated pdCpA.
R-methyl substituent was found to retain its chirality based on
NMR analysis. This implies that R-(S) is permissive for the
ribosome, although, at this stage, we could not completely
exclude the misincorporation of the preferable (R)-counterpart.40
Contrasting results were obtained for side chains at the â
position. At the â position, only the S configuration (â-(S)) was
accepted with moderate incorporation efficiency (37% ( 8%,
Figure 6A). However, the R configuration was almost rejected
(9% ( 3%, Figure 6A).
We then investigated the incorporation of R-(R)-methyl-â-
HPA at different codon sites. Escherichia coli DHFR mRNAs
containing the amber mutation at positions 10, 111, or 140 were
prepared and subjected to a gel-based amber suppression assay
under typical suppression conditions. To evaluate the effects
of the side chain, the incorporation of simple â-HPA was
simultaneously assessed under the same conditions, and Nap
was used as the typical positive control. As shown in Figure
6B, the â-HPA derivative R-(R)-methyl-â-HPA was clearly
(40) The optical purity of (S)-3-DMTr-oxy-2-methylpropionic acid, a precursor
of R-(S)-Me-â-HPA-pdCpA, was determined to be g95% ee by HPLC
analysis on a SUMICHIRAL OA-3100 column with 10 mM NH4OAc in
MeOH as eluent, suggesting that the contamination by the R enantiomer
can be non-negligible (e2.5%).
(41) Borek, E.; Clarke, H. T. J. Biol. Chem. 1938, 125, 479-494.
(42) Krueger, P. J. In The chemistry of the hydrazo, azo and azoxy groups;
Patai, A., Ed.; John Wiley: New York, 1975; pp 159-175.
(43) Rodnina, M. V.; Beringer, M.; Bieling, P. Biochem. Soc. Trans. 2005, 33,
493-498.
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Unexpected Preference of the E. coli Translation System
A R T I C L E S
mM potassium phosphate, 2.5 mM MgCl2, 6.5 mM Mg(OAc)2, 5 mM
ammonium acetate, pH 7.3) containing dC (6 nmol) as an internal
standard. The resulting solution was incubated at 37 °C, and the time-
course of the deacylation of the misacylated pdCpA was monitored by
HPLC. HPLC conditions: on a Wakosil 5C18 column (4.6 × 150 mm)
eluted with 0.1 M triethylammonium acetate buffer containing a 0-10%
acetonitrile linear gradient over 30 min (Gly, R-HAA, and â-HPA) or
0.05 M ammonium formate buffer containing a 0-10% acetonitrile
linear gradient over 40 min (â-Ala) as eluent, at a flow rate of 1 mL/
min, and with detection at 260 nm.
that the side chain at R-(R) causes a favorable anchoring effect
for substrate positioning and/or for the orientation of the reacting
groups, allowing efficient formation of the ester bond. It is also
interesting to note that the restrictions on the side-chain
modifications are loose. Only â-(R)-methyl-HPA was almost
rejected. This allows the preparation of unique library compo-
nents by diversification at R-(R), â-(S), and possibly at R-(S)
on main-chain-elongated scaffolds.
Conclusion
mRNA Templates. mRNA templates encoding E. coli DHFR and
oligopeptides were prepared by transcription (T7 MegaShortScript Kit,
Ambion) from a dsDNA template generated by PCR and purified with
the RNeasy MinElute Kit (Qiagen). For further details, see Supporting
Information.
The conlusion of this work is that, in contrast to â-amino
acid, the â-hydroxy acid that exists in the neutral OH state is a
rather good substrate of translation machinery. This automati-
cally indicates that it possesses sufficient activity in every respect
required for ribosomal chain-elongation reactions such as
nucleophilicity and binding to EF-Tu.44,45 At the same time, it
is also important to note that this does not allow any mechanistic
conclusion to be drawn about the poor reactivity of the â-amino
acid. In addition to the protonation-induced deactivation, there
can be other explanations based on RS-catalyzed editing, affinity
to EF-Tu, or inherent substrate selectivity of tRNA. A recent
report has even suggested that the ribosome enhances the rate
of PT reaction mainly by entropic gain, not by conventional
chemical catalysis.43 The next challenge is to find practical
factors governing the incorporation or rejection of main-chain
elongated substrates. Whatever the mechanistic details may be,
it is remarkable and even surprising that the less nucleophilic
â-hydroxy acid is a better substrate for the E. coli translation
system than the â-amino acid, at least when the chemically
Translation of E. coli DHFR and SDS-PAGE Analysis. Gel
electrophoresis and blotting were carried out on a model BE-250
electrophoresis apparatus (Biocraft Co., Ltd., Japan) and a Trans-Blot
SD Semi-Dry Transfer Cell (Bio-Rad Laboratories), respectively. The
reconstituted E. coli translation system48 (PureSystem-RF1, Classic I)
was purchased from Post Genome Co., Ltd. (Japan). Translation was
initiated by the addition of 2 µg of mRNA template for E. coli DHFR
with or without ∼5 µg of chemically misacylated tRNA (total reaction
volume, 11 µL). The reaction mixture was incubated at 37 °C for 60
min. The reaction solution was mixed with 27.5 µL of sample
loading buffer (125 mM Tris-HCl [pH 6.8], 4% [w/v] SDS, 20% [w/v]
glycerol, 0.002% [w/v] bromophenol blue, 10% [v/v] 2-mercaptoet-
hanol) and 16.5 µL of water. The resulting solution was incubated
at 95 °C for 5 min, and then 5 µL of this solution was applied
to 15% SDS-PAGE. Western blotting was carried out on a PVDF
membrane (Hybond-P, GE Healthcare). T7-tagged proteins were
visualized with horseradish-peroxidase-conjugated anti-T7 antibody
(Novagen) and ECL Plus Western Blotting Detection Reagent (GE
Healthcare). The relative yields of full-length DHFR were determined
after calibration with a set of serially diluted (0%, 5%, 10%, 20%,
40%, 60%, 80%, 100%) solutions of DHFR translated from dhfrwild
mRNA.
misacylated yeast tRNAPhe
is used as the vehicle for the
CUA
substrate. In other words, the E. coli translation machinery might
inherently be the system better suited to ester-bond formation
than to the natural-type amide-bond formation, as far as the
production of nonnatural products with elongated backbones is
concerned.46 These results raise the possibility of using â-HPA
and its derivatives to build a structurally diverse library with
the ribosomal decoding system. In view of the steric capacity
of the ribosome to accept â-substrates, further modification
thereof, that of tRNA, or pH control of the translation conditions
might allow the addition of â-amino acids to the repertoire of
efficiently decodable library components. Another possible
strategy involves chemical modifications that control the pKa
of the â-amino group, for example, by introducing an electron-
withdrawing group, such as a mono-, di-, or tri-fluoro moiety,
to the backbone or side chain to lower the pKa.47 Further work
is now underway to address these issues.
Translation of Oligopeptide and S-Tag-Based Assay. Translation
(total reaction volume, 10.6 µL; PureSystem-RF1) was initiated by the
addition of 2 µg of oligopepamber mRNA template with or without ∼4
µg of chemically misacylated tRNA. The reaction mixture was
incubated at 37 °C for 60 min. An S-tag-based suppression assay was
carried out using FRETWorks S-Tag Assay Kit (Novagen) according
to the manufacturer’s protocol. Briefly, the reaction solution (20 µL,
diluted 1/1000) was mixed with FRETWorks reaction mix (180 µL) in
a 96-well plate. After 5 min incubation, 20 µL of 10× stop solution
was added to the mixture. The fluorescence intensity of the resulting
solution was measured with a Wallac 1420 multilabel counter. The
relative yields of full-length oligopeptide were determined after
calibration with a set of serially diluted (0%, 5%, 10%, 20%, 40%,
60%, 80%, 100%) solutions of oligopeptide translated from oligopepY
mRNA.
Experimental Section
Chemically Misacylated Yeast tRNAPheCUA. The synthesis of
natural/nonnatural substrates, preparation of misacylated pdCpAs, and
enzymatic ligation with yeast tRNAPheCUA-CA37 are described in the
Supporting Information.
Hydrolysis of Misacylated pdCpA and HPLC Analysis. Misacy-
lated pdCpA (3 nmol) was dissolved in 30 µL of translation buffer (50
Purification and Mass Analysis of Oligopeptide. MALDI-TOF
mass spectra were measured on a Voyager Elite instrument (Applied
Biosystems) using R-cyano-4-hydroxycinnamic acid as the matrix.
Translation (PureSystem-RF1) was initiated by the addition of 8 µg of
the mRNA template (oligopepY or oligopepamber) with or without ∼16
µg of chemically misacylated tRNA (naphthylalanyl-tRNA) (total
reaction volume, 42.4 µL). The translation mixture was incubated at
37 °C for 60 min. To this mixture were added 45 µL of anti-FLAG
M2 affinity gel (Sigma) and 350 µL of lysis buffer (50 mM Tris-HCl
[pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20). The mixture
was gently inverted for 4 h at 4 °C, applied to a MicroSpin column
(44) LaRiviere, F. J.; Wolfson, A. D.; Uhlenbeck, O. C. Science 2001, 294,
165-168.
(45) Nakata, H.; Ohtsuki, T.; Abe, R.; Hohsaka, T.; Sisido, M. Anal. Biochem.
2006, 348, 321-323.
(46) Preliminary experiments showed that neither â-mercaptopropionic acid nor
R-mercaptoacetic acid could be incorporated. Thus, the sulfhydryl group
or the resultant thioester group seems to be not compatible with the present
translation conditions.
(48) Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa,
(47) Schlosser, M. Angew. Chem., Int. Ed. 1998, 110, 1496-1513.
K.; Ueda, T. Nat. Biotechnol. 2001, 19, 751-755.
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A R T I C L E S
Sando et al.
(GE Healthcare), and filtered by centrifugation. The agarose beads were
washed five times with 300 µL of prechilled TBS buffer and
resuspended in 170 (100 + 70) µL of elution buffer (0.1 M glycine
HCl, pH 3.5). The suspension was incubated with gentle shaking for 5
min at room temperature and filtered by centrifugation. To the filtrate
was added 17 µL of 0.5 M Tris-HCl (pH 7.4) containing 1.5 M NaCl,
and the resulting solution was dialyzed (Mini Dialysis Kit, 1 kDa cut
off; GE Healthcare) against a large volume of distilled water for 24 h
to purify the oligopeptide. The resulting solution was lyophilized and
dissolved in 5 µL of a 10 mg/mL solution of R-cyano-4-hydroxycin-
namic acid dissolved in a 1:1 mixture of water and acetonitrile
containing 0.3% trifluoroacetic acid. The resulting mixture (1-2 µL)
was spotted onto a MALDI plate, air-dried, and analyzed. For the
alkaline hydrolytic treatment, the lyophilized sample was dissolved in
5 µL of H2O and 3 µL of 25% ammonium hydroxide solution. The
resulting mixture was incubated at 37 °C for 24 h. After incubation,
the solution was dried under reduced pressure, dissolved in 2 µL of
the R-cyano-4-hydroxycinnamic acid matrix, and analyzed.
Acknowledgment. This paper is dedicated to Professor Isao
Saito on the occasion of his 65th birthday. We thank Dr. K.
Kanatani for his contribution in the early stages of this work.
We are grateful to Prof. T. Ueda, Dr. Y. Shimizu, and Dr. T.
Kanamori of the University of Tokyo for their advice and the
kind gift of the reconstituted translation system used in part of
this work. We also thank Dr. T. Hohsaka of JAIST and Dr. M.
Endo of Osaka University for their kind gifts of plasmid. This
work was partly supported by Grant-in-Aid no. 18350084 from
the Ministry of Education, Science, Sports, and Culture, Japan.
Supporting Information Available: General details, synthesis
of misacylated pdCpA, preparation of chemically misacylated
full-length tRNA, construction of expression template, and
Figures S1-S3. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA068033N
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6186 J. AM. CHEM. SOC. VOL. 129, NO. 19, 2007