In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy

Article abstract of DOI:10.1021/ja801602q

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe), ¹³C/¹⁵N-labeled p-methoxyphenylalanine (OMePhe), and ¹⁵N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF₃Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in ¹H- ¹⁵N HSQC, ¹H-¹³C HSQC, and ¹⁹F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF₃Phe mutants of an active site tyrosine inhibited binding; incorporating ¹⁵N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Full text of DOI:10.1021/ja801602q

Published on Web 06/25/2008
In vivo Incorporation of Unnatural Amino Acids to Probe
Structure, Dynamics, and Ligand Binding in a Large Protein
by Nuclear Magnetic Resonance Spectroscopy
Susan E. Cellitti,† David H. Jones,† Leanna Lagpacan,† Xueshi Hao,†
Qiong Zhang,† Huiyong Hu,† Scott M. Brittain,† Achim Brinker,† Jeremy Caldwell,†
Badry Bursulaya,† Glen Spraggon,† Ansgar Brock,† Youngha Ryu,‡ Tetsuo Uno,†
Peter G. Schultz,†,‡ and Bernhard H. Geierstanger*,†
Genomics Institute of the NoVartis Research Foundation, 10675 John Jay Hopkins DriVe, San
Diego, California 92121-1125, and Department of Chemistry and the Skaggs Institute for
Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
Received March 3, 2008; E-mail: bgeierst@gnf.org
Abstract: In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically
reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by
coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid
added to the media and the protein of interest with a TAG amber codon at the desired incorporation site.
To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic
acid (OCF3Phe), 13C/15N-labeled p-methoxyphenylalanine (OMePhe), and 15N-labeled o-nitrobenzyl-tyrosine
(oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase
domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for
OCF3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of
the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8
and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample,
were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples
were then used to study the binding of a tool compound. Chemical shift changes in 1H-15N HSQC, 1H-13C
HSQC, and 19F NMR spectra of the different single site mutants consistently identified the binding site and
the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF3Phe
mutants of an active site tyrosine inhibited binding; incorporating 15N-Tyr at this site through UV-cleavage
of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust
methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue
for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat
that can currently not be accomplished with any other method.
labeling (SAIL) amino acids,9 or selective isotope labeling of
methyl groups in perdeuterated proteins10,11 is very helpful for
Introduction
NMR spectroscopy is an established and very powerful
biophysical method to study the structure, dynamics, and
function of proteins.1–3 In principle, molecular processes such
as binding or conformational changes can be deciphered by
NMR with atomic resolution. However, the complexity of NMR
spectra of large proteins in practice often represents a formidable
challenge. Simplification of spectra by amino acid-type specific
isotope labeling4 including labeling with fluorinated analogs of
natural amino acids,5–8 by incorporation of stereoarray isotope
many applications and required for larger systems. Recently
developed methods for the in ViVo incorporation of unnatural
amino acids into proteins12–18 provide an unique opportunity
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† Novartis Research Foundation.
‡ The Scripps Research Institute.
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10.1021/ja801602q CCC: $40.75
2008 American Chemical Society

In vivo Incorporation of Unnatural Amino Acids
A R T I C L E S
Figure 1. Isotopically labeled unnatural amino acids O-methyl-phenylalanine (OMePhe), its trifluorinated analog OCF3Phe, and o-nitrobenzyl-tyrosine
(oNBTyr), which is readily converted into tyrosine upon UV illumination.23
to introduce NMR-active labels at a single designated amino
acid residue.19
enzyme catalysis, product release, and protein folding.21 The
unnatural amino acid labeling technique presented here has the
potential to extend NMR studies to many proteins that are
currently not amendable because of their complexity, size, or
physical characteristics.
Using an orthogonal amber tRNA/tRNA synthetase (tRNA/
RS) pair that specifically amino-acylates an amber tRNA with
the desired unnatural amino acid and incorporates it into a
protein at the amber nonsense codon UAG, any desired protein
residue can, at least in principle, be substituted in ViVo by an
unnatural amino acid. The incorporation of the unnatural amino
acid at the desired location instantly provides an “assignment”
for the NMR signal of the unnatural amino acid. Monitoring
the chemical shift change of a single resonance opens an avenue
for focused, site-directed screening for binders. This is particu-
larly interesting for drug development because it can reduce
the number of screening hits that bind to protein pockets of
little interest. Furthermore, unnatural amino acids incorporated
at different sites in multiple samples can be used to establish
binding within an active site or could be used to triangulate
binding of a small molecule or a biomacromolecule via NOE
measurements. Similarly, saturation transfer techniques that
utilize selective labeling strategies to study the structures of
protein-ligand complexes20 can utilize the specificity afforded
by unnatural amino acid incorporation to determine both the
protein residues and regions of the molecule that are in close
proximity. Protein conformational changes and dynamics can
also be monitored at specific sites of interest with minimal or
no perturbation of sequence. Mixtures of proteins labeled with
different unnatural amino acids can simplify the NMR charac-
terization of symmetric oligomers. Selectively incorporating
methyl labels can be very useful for examining the millisecond
time scales associated with many biological processes, such as
Previously, we have shown that protein amounts sufficient
for NMR studies can be produced in Escherichia coli using 15N-
labeled O-methyl-phenylalanine (OMePhe) (Figure 1).19 Be-
cause the yields were modest, one of the primary goals of the
current study was to improve the incorporation efficiency and
consequently increase protein yields. This was accomplished
by constructing a plasmid that overexpresses the RS specific
for the unnatural amino acid at the time of induction. The second
goal was to expand the number of NMR-active unnatural amino
acids. Producing 15N and 13C-labeled OMePhe was an obvious
first step (Figure 1). Developing an orthogonal tRNA/RS pair
for the incorporation of a trifluorinated analog was our second
choice. Fluorine represents an attractive NMR label5–8 with high
intrinsic sensitivity, 100% natural abundance, and the absence
of any natural background. The chemical shift of 19F is
extremely sensitive to the local environment and can detect
changes in van der Waals contacts, electrostatic fields and
hydrogen bonding. In contrast to Mehl and co-workers who
recently selected a specific tRNA/RS pair for trifluoromethyl-
phenylalanine and demonstrated its utility for 19F protein NMR
studies,22 we opted to develop such a system for OCF3Phe
(Figure 1) because of its structural similarity to OMePhe. As a
third unnatural amino acid, we tested the incorporation of 15N-
labeled o-nitrobenzyl-tyrosine (oNBTyr) (Figure 1). It has been
demonstrated that UV-photocleavage of this unnatural amino
acid after incorporation converts oNBTyr back to the natural
amino acid tyrosine.23 In contrast to the other unnatural amino
(16) Xie, J.; Schultz, P. G. Curr. Opin. Chem. Biol. 2005, 9, 548–554.
(17) Xie, J.; Schultz, P. G. Methods 2005, 36, 227–238.
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2007, 4, 239–244.
(21) Skrynnikov, N. R.; Mulder, F. A. A.; Hon, B.; Dahlquist, F. W.; Kay,
L. E. J. Am. Chem. Soc. 2001, 123, 4556–4566.
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(19) Deiters, A.; Geierstanger, B. H.; Schultz, P. G. ChemBioChem 2005,
6, 55–58.
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P. J.; Beutel, B. A. J. Am. Chem. Soc. 2004, 126, 2390–2398.
(23) Deiters, A.; Groff, D.; Ryu, Y.; Xie, J.; Schultz, P. G. Angew. Chem.,
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9
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A R T I C L E S
Cellitti et al.
Scheme 1a
a Conditions: (a) (tBuOCO)2O, NEt3, H2O/1,4-dioxane (1:1 v/v), 23 °C, 16 h, 92%; (b) 13CH3I, K2CO3, DMF, 0-23 °C, 97%; (c) TFA/DCM (1:4 v/v),
23 °C; (d) NaOH, H2O/MeOH (1:1 v/v), 23 °C, 41% (2 steps).
Scheme 2a
a Conditions: (a) CuSO45H2O, NaOH, H2O, 23 °C; (b) 2-nitrobenzyl chloride, NaOH, H2O/MeOH (1:1 v/v), DMF, 23 °C, 46% (2 steps); (c) 1 M HCl,
23 °C, 83%.
labeled oNBTyr were prepared from 15N-labeled L-tyrosine (Cam-
acids, photocaged tyrosine facilitates the introduction of an NMR
bridge Isotopes Laboratory) according to Schemes 1 and 2,
respectively. Tool compound 17 was synthesized according to
Scheme 3 (Vide infra). Deuterated imidazole and D2O were obtained
from Cambridge Isotopes Laboratory and TCEP was obtained from
Pierce. All other chemicals were obtained from Sigma.
label at specific tyrosine residues without altering the protein
sequence.
The three different NMR-active unnatural amino acids were
used to study the binding of a small molecule ligand to the
thioesterase domain of fatty acid synthase (FAS-TE), a 33 kDa
protein of pharmaceutical interest. Fatty acid synthase (FAS)
is a large, multidomain enzyme essential for the synthesis of
long-chain fatty acids.24 Because the sequence and architecture
of FAS differ significantly between bacteria and mammals,
bacterial FAS has long been considered a valuable target for
the development of novel antibiotics (see ref 25 and references
herein). In humans, FAS is overexpressed in many cancers26
and is a drug target for obesity and related diseases.27 Orlistat,
an approved obesity drug, exhibits antitumor activity by
inhibiting FAS-TE.28,29 The molecular details of orlistat’s
interactions with FAS-TE have only been revealed in a recent
X-ray crystallographic study.30 Here we use a more soluble tool
compound to evaluate the utility of unnatural amino acids for
the characterization of protein-ligand interactions. Successful
incorporation of three different NMR-active unnatural amino
acids at 11 different positions around the proposed binding site
in FAS-TE demonstrates the general utility of the approach to
studies of protein structure, dynamics, function, and binding of
small molecules in particular.
Plasmids for the Expression of FAS-TE and Generation of
Mutants. The coding sequence of our model protein, FAS-TE, was
inserted into pMH4 (JCSG) using the PIPE cloning method.31 TAG
codons were mutated in at 11 specific sites using the same cloning
technique. The sites of incorporation were chosen based on the
solvent accessibility of the side chain in the FAS-TE apo-structure
(1XKT.pdb).32 To lower termination efficiency and subsequently
reduce truncation at the TAG stop codon, the codon of the residue
C-terminal of the incorporation site was optimized33 for Tyr-2351
(Arg-2352: CGG to AGG, silent mutation) and for Leu-2222 (Leu-
2223: CTG to ATT, resulting in a Leu-2223-Ile mutation). Cys-
2359 was mutated to Ser to prevent disulphide mediated aggregation.
Plasmids for the Incorporation of OMePhe and oNBTyr and
Improving the Expression Plasmid. Initial incorporation tests were
performed with the previously described pSUP-OMePhe-3TRN
plasmid.13 This plasmid contains three copies of an amber tRNA
and the mutant Methanococcus jannaschii tyrosyl-tRNA synthetase
selected for OMePhe under the control of a glnS promotor. To
coexpress the tRNA synthetase and therefore enhance amino
acylation activity during protein production, we modified the
plasmid as follows: For each synthetase of interest, the coding
sequence was inserted into pMH4 by PIPE cloning.31 The resulting
plasmid was then used as template for a standard PCR amplification
using the oligos KpnRS_F (5′-GGAGGTACCCAATTATGA-
CAACTTGACGGCTACATC-3′) and SacRS_R (5′-GGAAC-
GAGCTCACAAACAACAGATAAAACGAAAGGCCC-3′). The
PCR product, containing the entire arabinose operon, was then
prepared by restriction digest and inserted by ligation between the
KpnI and SacI sites in the pSUP-RS-3TRN vector13 with the same
synthetase gene. The result is pSUPAR3-RS containing all of the
elements of the pSUP-RS-3TRN vector plus an additional arabinose-
inducible copy of the synthetase (Figure 2).
Materials and Methods
Unnatural Amino Acids, Tool Compound, and Other
Chemicals. OCF3-DL-Phe was purchased from JRD Fluorochemi-
cals (Leatherhead, Surrey, UK) and used without further purifica-
tion. OMe-L-Phe was purchased from Sigma-Aldrich and used
without further purification. 13C/15N-labeled OMePhe and 15N-
(24) Jayakumar, A.; Tai, M. H.; Huang, W. Y.; al-Feel, W.; Hsu, M.; Abu-
Elheiga, L.; Chirala, S. S.; Wakil, S. J. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 8695–9.
Plasmids and Libraries for the Selection of a OCF3Phe-
Specific tRNA Synthetase. All plasmids used in the selection of
a OCF3Phe-specific tRNA synthetase were described previ-
(25) White, S. W.; Zheng, J.; Zhang, Y. M.; Rock, C. H. Annu. ReV.
Biochem. 2005, 74, 791–831.
(26) Kuhajda, F. P. Cancer Res. 2006, 66, 5977–5980.
(27) Ronnett, G. V.; Kleman, A. M.; Kim, E. K.; Landree, L. E.; Tu, Y.
Obesity 2006, 14 (14, Suppl 5), 201S–207S.
(28) Kridel, S. J.; Axelrod, F.; Rozenkrantz, N.; Smith, J. W. Cancer Res.
2004, 64, 2070–2075.
(31) Klock, H. E.; Koesema, E. J.; Knuth, M. W.; Lesley, S. A. Proteins
2008, 71, 982–994.
(32) Chakravarty, B.; Gu, Z.; Chirala, S. S.; Wakil, S. J.; Quiocho, F. A.
Proc. Nat. Acad. Sci. U.S.A. 2004, 101, 15567–15572.
(33) Brown, C. M.; Stockwell, P. A.; Trotman, C. N.; Tate, W. P. Nucleic
Acids Res. 1990, 18, 6339–6345.
(29) Knowles, L. M.; Axelrod, F.; Browne, C. D.; Smith, J. W. J. Biol.
Chem. 2004, 279, 30540–30545.
(30) Pemble, C. W.; Johnson, L. C.; Kridel, S. J.; Lowther, W. T. Nat.
Struct. Mol. Biol. 2007, 14, 704–709.
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In vivo Incorporation of Unnatural Amino Acids
A R T I C L E S
Scheme 3a
a Conditions: (a)3,4-(methylenedioxy)phenylmagnesium bromide, THF/toluene (1:1 v/v),-78 to 23 °C, 98%; (b) 1 M HCl, THF, 70 °C; (c)
trifluoromethanesulfonyl azide, CuSO4, K2CO3, H2O/MeOH, 0-23 °C, 42% (2 steps); (d) 4-nitrobenzene-1-sulfonyl chloride, pyridine, cat. DMAP, DCM,
23 °C, 55%; (e) cyclohexanamine, NMP, 80 °C; (f) 2,4-difluorobenzoyl chloride, DIEA, DCM, 23 °C, 65%; (g) H2, 5% Pd/C, MeOH, 23 °C; (h)
n-butylisocyanate, DIEA, DCM, 23 °C, 28% (2 steps).
Figure 2. pSUPAR3 plasmid. Plasmid contains two copies of the amino-acyl tRNA synthetase specific for the unnatural amino acid (Uaa-RS), one under
the control of a constitutively on glnS promoter and the second under the control of an arabinose-inducible ara promoter. The plasmid also encodes three
copies of the unnatural amino acid specific amber tRNA with a proK promoter, a p15A origin and a chloramphenicol CmR resistance marker.
ously.16,34 pREP2-YC-JYCUA encodes the mutated tRNATyr
from M. jannaschii, a chloramphenicol resistance gene containing
Selection of a Novel OCF3Phe-Specific tRNA Synthetase. In
the first round of selection, frozen aliquots, equivalent to ∼1010
cfu, of DH10B cells transformed with pREP2-YC-JYCUA and each
of the pBK library plasmids were plated on GMML-Agar supple-
mented with 0.04% glucose; 50 µg/mL kanamycin; 0.25 mM
OCF3Phe; and 50 µg/mL chloramphenicol or on LB-Agar with 50
µg/mL kanamycin; 0.5 mM OCF3Phe; and 50 µg/mL chloram-
phenicol. After 3 days, cells were scraped from the plates and the
plasmids purified by Spin Miniprep Kit (Qiagen). The pREP2 and
pBK plasmids were separated by gel electrophoresis on a 1%
agarose gel, and the pBK plasmid pools were repurified by Minelute
Gel Extraction Kit (Qiagen). For negative selections, the pBK pools
were transformed by electroporation into HK100 cells (JCSG;
derived from Genehogs by Invitrogen) harboring the pNEG plasmid
and plated on LB-Agar supplemented with 50 µg/mL kanamycin,
100 µg/mL ampicillin, and 0.2% arabinose. After 12-14 h of
growth, the pBK plasmid pools were isolated as before and
transformed into HK100 cells with the pREP2 plasmid. After a
total of four positive rounds (with 50, 50, 75, and 100 µg/mL
chloramphenicol, with 0.25, 0.25, 0.5, and 0.5 mM OCF3Phe, and
with all defined media plates after the first round) and three negative
CUA
a TAG stop codon, and a tetracycline resistance gene. pNEG
encodes the mutated tRNATyr
barnase with three TAG stop
CUA,
codons in the coding sequence, and ꢀ-lactamase. pBK-MJYRS-L1
(positions Tyr32, Leu65, His70, Gln155, Asp158, and Leu162
randomized), pBK-MJYRS-L234 (positions Tyr32, Leu65, Phe108,
Gln109, Asp158, and Leu162 randomized), and pBK-MJYRS-
L3D35 (positions Tyr32, Leu65, His70, Phe108, Gln109, Gln155,
Asp158, Ile159, and Leu162 randomized) encode the tyrosyl tRNA
synthetase (aa RS) from M. jannaschii with different positions of
the active site randomized to all 20 amino acids, as previously
described.34,35 pLeiZ encodes the Z-domain protein with a TAG
codon at position 7 and C-terminal His6-tag, the MjtRNATyr
and ꢀ-lactamase.36
,
CUA
(34) Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science 2001, 292,
498–500.
(35) Schultz, K. C.; Supekova, L.; Ryu, Y. H.; Xie, J. M.; Perera, R.;
Schultz, P. G. J. Am. Chem. Soc. 2006, 128, 13984–13985.
(36) Wang, L.; Zhang, Z.; Brock, A.; Schultz, P. G. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 56–61.
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A R T I C L E S
Cellitti et al.
Table 1. Incorporation of Three Unnatural Amino Acids into
rounds, colonies were picked from the final positive round for
further study. Following the positive selection recipe, a total of
296 colonies were replica plated onto agar plates either without
OCF3Phe but with 20, 35, or 50 µg/mL chloramphenicol; with 0.5
mM OCF3Phe and 100, 125, or 150 µg/mL chloramphenicol; or
with 1 mM OCF3Phe and 100, 125, or 150 µg/mL chloramphenicol.
The plasmids from 37 of these colonies that showed high growth
with unnatural amino acid and poor growth without it were isolated
and sequenced, revealing 14 novel sequences (Table S1 and S2).
Five clones (A6, B7, B10, F6 and H4) were chosen for further
evaluation based on the results of pooled replica plates and the
number of occurrences of the clone.
FAS-TE at 11 Positions Using pSUPAR3 and 2.5 mM 13C/15
N
-Labeled OMePhe, 1 mM OCF3Phe, and 0.5 mM 15N-Labeled
oNBTyra
FAS-TE
Mutant
Yield per 50 mL of culture [mg/50 mL] NMR sample concentration [mM]
OMePhe
OCF3Phe
oNBTyr
OMePhe OCF3Phe oNBTyr
Leu-2222
Thr-2255
Tyr-2307
Tyr-2343
Tyr-2347
Tyr-2351
Gln-2373
Phe-2375
His-2408
Thr-2450
Tyr-2454
2.7
2.2
1.6
0.47
1.3
1.7
0.67
2.4
2.0
0.84
2.1
2.2
3.1
2.0
2.2
2.3
1.4
0.42
1.8
2.7
2.5
1.2
1.2
2.1
0.94
1.9
2.5
1.9
0.25
4.5
5.0
1.9
7.2
0.15
0.12
0.12
0.17
0.11
0.12
0.11
0.080
0.10
0.13
0.27
0.16
0.16
0.11
0.14
0.052
0.12
0.088
0.051
0.072
0.095
0.055
0.096
0.11
0.13
0.077
0.034
0.096
0.15
0.14
0.066
Test Incorporation of OCF3Phe into Z-Domain Protein
and Evaluation of Misincorporation. HK100 cells cotransformed
with pLeiZ and each of the five pBK-OCF3Phe-RS were grown in
50 mL cultures of TB supplemented with 50 µg/mL kanamycin,
100 µg/mL ampicillin, and with or without 1 mM OCF3Phe. Cells
were harvested after 5 h induction with 1 mM IPTG at 30 °C.
Lysates were prepared in 6 M guanidine by sonication and clarified
by centrifugation at 20 000g for 20 min. The His-tagged proteins
from each culture were purified by Ni-NTA (Qiagen) columns
according to the manufacturer’s protocol. Denaturant was removed
by PD-10 columns (GE Healthcare). The protein samples were next
evaluated by SDS-PAGE, Bradford assay (Pierce), and LC-ESI-
MS (Supplemental Figures S2 and S3, Supporting Information).
To test for miscorporation of natural amino acids at the TAG
incorporation site, protein samples of Z-domain expressed in the
absence or presence of OCF3Phe with the five evolved OCF3Phe-
RS were digested with trypsin and subjected to LC-ESI-MS to
evaluate the presence of the N-terminal peptide TSVDNXINK,
where X represents the mutated position. The peptide masses for
incorporation of OCF3Phe, Tyr, Phe, and Trp were all monitored
in MS experiments to evaluate misincorporation levels of the natural
aromatic amino acids. The Z-domain protein produced with
OCF3Phe-RS clone A6 in the presence of 1 mM OCF3Phe was
used to verify the sequence of the peptide by MS-MS. In a separate
experiment, the incorporation fidelity of the OCF3Phe RS (clone
F6) was further investigated at the Thr-26 site of FKBP1237 by
multiple-reaction monitoring as described previously.38 The ho-
mogeneity of the protein was found to be better than 99.5%.
0.099
0.11
a Yields after purification are given in milligram per 50 mL E.coli
culture. The concentrations for the NMR samples shown in Figures 4, 5,
and 6, respectively, are also listed.
Expression of FAS-TE Mutants Selectively Labeled with
15N-Tyrosine. The tyrosine residues of wild-type and mutant FAS-
TE were selectively labeled using the following protocol: 20 mL
of an overnight TB culture was added per 1 L M9 media (Sigma)
supplemented with 0.5% glycerol and 0.1 mM FeCl3. Thirty minutes
prior to induction a sterile 100 mL solution containing 100 mg 15N-
labeled tyrosine; 500 mg each of glutamic acid, aspartic acid,
glutamine, and asparagine; 300 mg each of alanine, valine,
isoleucine, leucine, tryptophan, phenylalanine, serine, and glycine;
and 200 mg of each of the remaining amino acids was added. The
culture was induced as described above and allowed to proceed
for approximately one-fifth of the normal expression time to
minimize scrambling.
NMR Sample Preparation and Data Collection. As a lock
solvent, 50 µL of D2O were added to 500 µL of protein solution
resulting in a final buffer composition of 18 mM d-imidazole, 91
mM NaCl, 0.45 mM TCEP, pH 6.5. The final protein concentrations
of all NMR samples are given in Table 1. Aliquots of the tool
compound were added from a 50 mM stock in deuterated DMSO
to a final concentration of 1.4 mol equiv unless mentioned
otherwise. The final DMSO concentration was between 0.10% and
0.76% v.v. for all samples but typically less than 0.5% v.v. resulting
in negligible chemical shift changes. All spectra were recorded at
300 K. 19F spectra were recorded on an Avance 400 MHz
Expression of FAS-TE Mutants Containing OMePhe, OCF3-
Phe, and oNBTyr. OCF3Phe-RS clone F6 was inserted into the
pSUPAR3 plasmid. For each mutant, the expression plasmid and
pSUPAR3-OMePhe, pSUPAR3-OCF3Phe, or pSUPAR3-oNBTyr
plasmid were cotransformed into HK100 cells. Multiple single
colonies were picked for growth in 50 mL TB cultures (in 250 mL
shaker flasks) with 100 µg/mL ampicillin and 35 µg/mL chloram-
phenicol. At OD595 ) 1.0, cultures were moved to 30 °C and
supplemented with 2.5 mM 13C/15N-labeled OMePhe, 1 mM
OCF3Phe (both from 0.5 M stock solutions in 1 N HCl) or 0.5
mM 15N-labeled oNBTyr (from a fresh 0.5 M stock in DMSO).
Thirty minutes later, expression was induced by addition of 0.2%
arabinose. After 20 h, cells were harvested. Lysates were prepared
by sonication, clarified by centrifugation, and purified by Ni-NTA
under native conditions according to the manufacturer’s protocol.
Protein samples were evaluated by SDS-PAGE, Bradford assay,
and ESI-MS before being exchanged into NMR buffer (20 mM
deuterated imidazole, 100 mM NaCl, 0.5 mM TCEP, pH 6.5) via
PD-10 column and concentrated using Amicon-ultra units (Milli-
pore). Expression yields after purification are given in Table 1.
Photocleaveage of oNBTyr was accomplished by exposing the
NMR samples for 10 min to UV light generated by an Oriel 500
W Hg light source (Newport Corporation; Stratford, CT).
1
instrument (Bruker Biospin, Billerica, MA) equipped with a H/
13C/19F/31P-QNP-cryoprobe. Spectra were typically recorded with
1024 scans, a recycle delay of 2 s, 8192 complex data points with
a sweep width of 20 ppm. Proton decoupling was accomplished
using Waltz-16. All other spectra were recorded on an Avance 600
MHz instrument equipped with a 1H/13C/15N-TXI-cryoprobe.
1H-13C HSQC39 were typically recorded with 32 scans, 128 t1
experiments at 300 K using a spectral width of 50 ppm in the carbon
dimension and 13.97 ppm in the proton dimension. Similarly,
1H-15N HSQC spectra40 were recorded using 48 scans, 256 t1
experiments using a spectral width of 32 and 13.97 ppm in the
nitrogen and proton dimension, respectively.
Results
Improving Incorporation Efficiency. A first incorporation test
of OMePhe into the thioesterase domain of fatty acid synthase
(FAS-TE) was performed in E. coli with the previously
described mutant M. jannaschii tyrosyl tRNA/tRNA synthetase
(tRNA/RS) pair encoded by the pSUP-OMePhe-6TRN plas-
mid.13 Fifty milliliter bacterial cultures were grown in rich media
(37) Michnick, S. W.; Rosen, M. K.; Wandless, T. J.; Karplus, M.;
Schreiber, S. L. Science 1991, 252, 836–839.
(38) Chen, S.; Schultz, P. G.; Brock, A. J. Mol. Biol. 2007, 371, 112–122.
(39) Davis, A. L.; Keeler, J.; Laue, E. D.; Moskau, D. J. Magn. Reson.
1992, 98, 207–216.
(40) Mori, S.; Abeygunawardana, C.; Johnson, M. O.; Vanzijl, P. C. M. J.
Magn. Reson. Series B 1995, 108, 94–98.
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In vivo Incorporation of Unnatural Amino Acids
A R T I C L E S
Figure 3. Incorporation of unnatural amino acids preserves structure of FAS-TE. 1H-15N HSQC of (A) ”reverse-labeled” (unlabeled oNBTyr incorporated
into uniformly 15N-labeled protein) FAS-TE Tyr-2454-oNBTyr before (blue) and after UV cleavage (red); (B) the UV-cleaved sample (red) overlaid with
uniformly 15N-labeled wild-type FAS-TE (black); (C) Overlay of wild-type protein before (black) and after addition of tool compound (green); and (D)
overlay of reverse labeled and UV-cleaved Tyr-2454-oNBTyr before (blue) and after addition of tool compound (red).
at OMePhe concentrations between 2.5 and 30 mM (between
25 and 300 mg per 50 mL) (Figure S1A and B, Supporting
Information). Only between ∼0.4 and 1.2 mg of full-length
protein was produced, respectively, compared to ∼3.8 mg for
wild-type protein produced under the same conditions. The
lower yield is caused by a high-rate of translation termination
at the UAG amber codon as large amounts of truncated FAS-
TE were detected in the insoluble fraction by SDS-gel and MS
analysis. To increase the concentration and activity of the mutant
tRNA synthetase, we constructed a plasmid pSUPAR3 that
features an additional RS gene that is under the control of an
arabinose inducible promoter (Figure 2). Simultaneous over-
expression of RS and of the target protein dramatically reduced
truncation and enhanced production of full-length FAS-TE
(Figure S1C and D, Supporting Information). Optimization of
the expression conditions showed that growth in TB media and
induction at 30 °C for 20 h further increased yields of both
wild-type and mutant proteins. Incorporation tests with the
pSUPAR3 plasmid at different concentrations of OMePhe
indicated saturation of protein production above 5 mM, sug-
gesting that unnatural amino acid and synthetase are not limiting
under these conditions. Comparisons of different versions of
pSUPAR3 revealed that expression of full-length FAS-TE Tyr-
2454-OMePhe was maximized by having 3 copies of the tRNA
(vs 1 or 6) and both the inducible and the noninducible copies
of the synthetase together (vs either copy alone). Changing the
number of tRNA copies did not enhance yields suggesting that
additional factors, that will be optimized in the future, are
limiting to achieve wild-type yield. Using the pSUPAR3-
OMePhe plasmid and only 2.5 mM OMePhe in 50 mL TB
media, mutant FAS-TE with a single OMePhe incorporated can
currently be produced with yields sufficient for a single 0.1 mM
NMR sample. Subsequently, existing orthogonal tRNA syn-
thetase for oNBTyr and a newly evolved tRNA synthetase for
OCF3Phe described below were also transferred to the pSU-
PAR3 plasmid and successfully used for protein expression.
Expanding the Number of NMR-Active Unnatural Amino
Acids. Gram quantities of isotope labeled analogs of unnatural
amino acids, namely a 13C/15N-labeled analog of OMePhe and
a
15N-labeled analog of oNBTyr (Figure 1) were synthesized.
13C/15N-labeled OMePhe 4 was prepared according to Scheme
1 whereas 15N-labeled oNBTyr 8 was prepared according to
9
J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008 9273

A R T I C L E S
Cellitti et al.
1
Figure 4. 1H-13C NMR spectra of FAS-TE mutants with 13C/15N -labeled OMePhe incorporated at 11 different positions: H-13C HSQC spectra before
(blue) and after addition of tool compound (red). With the exception of the compound complex of Tyr-2343-OMePhe, all methoxy cross peaks of the
unnatural amino acid have chemical shifts between 56.5 and 54.0 ppm for carbon and 3.85 and 3.35 ppm for protons. For Tyr-2343-OMePhe all observable
peaks are shown. These include natural abundance peaks of TCEP, residual glycerol from concentration devices, residual TRIS from the purification buffer
and other small molecule components of the NMR buffer. These peaks are also observed for all other mutants for which only the boxed region in the
Tyr-2343-OMePhe mutant spectrum is shown.
Scheme 2 following procedures for the synthesis of o-benzyl-
L-tyrosine ether.41
(5JHF < 1 Hz, data not shown) should experience minimal
fluorine-proton relaxation resulting in high intrinsic sensitivity
for applications in large proteins, and (c) the tRNA/RS pair
specific for OMePhe should be a good starting point for the
evolution of a OCF3Phe-specific RS. Initial tests to incorporate
OCF3Phe into proteins using the OMePhe-specific tRNA/RS
pair failed and demonstrate the high selectivity of tRNA
synthetases for their substrates which is consistent with previous
suggestions that a CF3 group should be bulkier and more
hydrophobic than a CH3 group.42
In addition, an incorporation system for a fluorinated un-
natural amino acid was developed. Fluorinated analogs of natural
amino acids, especially of Phe, Tyr, and Trp, can be incorporated
into proteins at all respective aromatic residues and used for
protein NMR studies.5–8 A tRNA/RS pair for the site-specific
in ViVo incorporation of a fluorinated unnatural amino has so
far been developed for only trifluoromethyl-phenylalanine.22
Independently, we chose to develop such a system for OCF3Phe
(Figure 1) because (a) its structure is a conservative modification
of the unnatural amino acid OMePhe that can be introduced
without adverse effects on the structure of our test protein (see
below), (b) three fluorine atoms virtually decoupled from a ring
Randomizing amino acid residues in M. jannaschii TyrRS
that are in contact with the substrate tyrosine side chain as
illustrated in the X-ray structure43 enabled the selection of a
RS specific for OMePhe.34 We successfully used similar libraries
(41) Bodanzky, N.; Bodanzky, A. The Practice of Peptide Synthesis, 2nd
(42) Budisa, N.; Pipitone, O.; Siwanowicz, I.; Rubini, M.; Pal, P. P.; Holak,
ed.; Springer Verlag: Berlin-Heidelberg, 1994.
T. A.; Gelmi, M. L. Chem. BiodiVers. 2004, 1, 1465–1475.
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In vivo Incorporation of Unnatural Amino Acids
A R T I C L E S
performed with a FAS-TE binder 17 that was discovered in a
high throughput screen (Caldwell et al., unpublished results).
Compound 17was synthesized according to Scheme 3. Briefly,
Garner’s aldehyde 9 was treated with a Grignard reagent,
affording benzyl alcohol 10 quantitatively, followed by the
global deprotecion by aqueous HCl. The amino group of 11
was masked as azide by trifluoromethanesulfonyl azide; the
configuration of the stereo center of the amino group was
retained under the condition.44 The primary hydroxyl group of
12 was selectively converted to the nosylate, and then to the
cyclohexylamino group by nucleophilic displacement. The
resulting intermediate, 14, was elaborated to the final compound,
17, by following the sequence of benzoylation, azide reduction,
and ureation. Although we were unable to determine the
diastereo selectivity of the first reaction (9 to 10), the 1H NMR
and LC-MS analysis of 13 indicated that 13 comprises mainly
one diastereomer. Because the synthesis started with homochiral
aldehyde 9 and no steps were considered to involve racemiza-
tion, we assume the final compound, 17, also comprises mainly
one species regarding the benzilic and homobenzylic positions.
Incorporation of OMePhe, OCF3Phe, and oNBTyr into 11
FAS-TE Mutant Proteins. Using pSUPAR3 plasmids, 11 FAS-
TE mutants were expressed with three unnatural amino acids
substituted at single sites. Positions Leu-2222, Thr-2255, Tyr-
2307, Tyr-2343, Gln-2373, Phe-2375, His-2408, and Thr-2450
were selected in order to place the unnatural amino acid in
solvent exposed positions surrounding the active site. Tyr-2347,
Tyr-2351 and Tyr-2454 were selected as additional incorporation
sites to probe adjacent protein regions that are disordered in
the crystal structures of apoprotein32 and the orlistat complex.30
Cells transformed with an expression plasmid for each mutant
and pSUPAR3-OMePhe, pSUPAR3-OCF3Phe or pSUPAR3-
oNBTyr were fed the respective unnatural amino acid and
induced to express the model proteins as described in Materials
and Methods. Each mutant was purified using Ni-affinity and
size-exclusion chromatography. The average yield of purified
protein was ∼2 mg per 50 mL culture (Table 1). With the
exception of Gln-2373-OMePhe, Gln-2373-OCF3Phe, Tyr-2307-
oNBTyr, Tyr-2347-oNBTyr, Tyr-2351-oNBTyr, and Gln-2373-
oNBTyr, purified protein from only one 50 mL culture was
transferred to NMR buffer resulting in NMR samples of
typically 0.1 mM in concentration (total volume 0.55 mL). The
predicted molecular weight of each mutant protein was con-
firmed by ESI-MS. Generally, there is little difference in the
incorporation yield for each of the three unnatural amino acids
at the same incorporation site. However, protein yields of the
different mutants varied significantly possibly because of
differences in incorporation efficiencies and effects on protein
stability during expression and purification.
Figure 5. 19F NMR spectra of FAS-TE mutants with OCF3Phe incorporated
at 11 different positions. Spectra in blue are of the uncomplexed protein
while the spectra in red were recorded after the addition of tool compound.
Significant line broadened, presumably because of conformational exchange,
is observed for some but not all residues. For comparison, a spectrum of a
0.5 mM sample of OCF3Phe is shown.
to select a mutant TyrRS specific for OCF3Phe and illustrate
that this RS selectively and specifically incorporates OCF3Phe
into Z-domain protein, FKBP12 and FAS-TE. Three libraries
of mutants of the M. jannaschii tyrosyl-tRNA synthetase were
subjected to alternating rounds of positive and negative selection
in order to isolate clones that specifically recognized OCF3Phe
and not any natural amino acids. Five promising RS clones,
A6 (Val32, Ala65, Gln108, Trp109, Ala158, Lys162), B7
(Val32, Ala108, Trp109, Gly158, Gln162), B10 (Ala32, Ala65,
Trp108, Met109, Gly158, Asn159), F6 (Ala32, Ser65, Gln108,
Ala109, Ala158, Tyr162) and H4 (Ile26, Val32, Gly65, His108,
Tyr109, Ala158A, His162) (Tables S1 and S2, Supporting
Information), were used to express Z-domain protein. In all five
cases, the Z-domain was expressed only in the presence of
OCF3Phe, as measured by SDS-PAGE and LC-MS (Figure S2,
Supporting Information). The expressed protein was confirmed
to contain OCF3Phe at the expected position by peptide
sequencing. Based on LC-ESI-MS, the ratio of expressed
protein containing a correct OCF3Phe amino acid compared to
a misincorporated Tyr, Phe, or Trp was greater than 300:1 for
two of the amino acyl-RS clones, A6 and F6 (Figure S3,
Supporting Information). Clone F6 was subsequently chosen
for future use because it consistently produced higher expression
yields.
Effects of Unnatural Amino Acid Incorporation on Pro-
tein Structure. NMR spectra of FAS-TE mutants individually
labeled at 11 positions with three different unnatural amino acids
were recorded in the absence and presence of a FAS-TE binding
tool compound as described below. Individual incorporation of
an unnatural amino acid does not grossly perturb the structure
of any of these FAS-TE mutants as indicated by only small
changes in the 1H NMR spectra (Figures S4, S5 and S6,
Supporting Information). All mutants retain well dispersed 1D
1H NMR spectra indicative of well folded proteins. For some
but not all proteins, structural integrity was further confirmed
FAS-TE Binding Tool Compound. Because orlistat is very
insoluble in aqueous solutions, our binding experiments were
(43) Kobayashi, T.; Nureki, O.; Ishitani, R.; Yaremchuk, A.; Tukalo, M.;
Cusack, S.; Sakamoto, K.; Yokoyama, S. Nat. Struct. Biol. 2003, 10,
425–432.
(44) Alper, P. B.; Hung, S. C.; Wong, C. H. Tetrahedron Lett. 1996, 37,
6029–6032.
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A R T I C L E S
Cellitti et al.
Figure 6. 1H-15N NMR spectra of FAS-TE mutants with 15N-labeled oNBTyr incorporated at 11 different positions: (A) 1H-15N HSQC spectra before
1
(black) and after UV-cleavage (blue), and after addition of tool compound (red). (B) H-15N HSQC spectra of wild-type FAS-TE selectively labeled with
15N-tyrosine before (blue) and after addition of tool compound (red). Spectra of individually 15N-oNBTyr labeled mutants facilitated signal assignments as
indicated by residue numbers.
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9276 J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008

In vivo Incorporation of Unnatural Amino Acids
A R T I C L E S
1
by H-15N HSQC spectra of uniformly 15N-labeled mutant
protein expressed in the presence of the unlabeled unnatural
amino acid (Figure 3 and Figure S7, Supporting Information).
For example, the ”reverse-labeled” (15N-labeled at all nitrogens
except for oNBTyr) Tyr-2454-oNBTyr mutant differences in
the amide cross peaks are restricted to a few resonances (Figure
3A) as would be expected for any well tolerated single site
mutation. Upon UV cleavage, oNBTyr is converted to Tyr and
as expected the 1H-15N HSQC spectrum becomes identical to
that of wild-type FAS-TE (Figure 3B). The resonance of Tyr-
2454 is, as discussed below, exchange broadened and conse-
quently not visible as an additional peak in the wild-type
spectrum. Both wild-type and reverse-labeled Tyr-2454-oNBTyr
show identical chemical shift changes upon tool compound
binding. Furthermore, a Tyr-2454-oNBTyr (unlabeled) mutant
sample selectively labeled with 15N-tyrosine gives rise to a
1H-15N spectrum identical to a 15N-tyrosine wild-type protein
sample (Figure S8, Supporting Information). Additional data
for reverse labeled samples are shown in Figure S7 (Supporting
Information) and all support the conclusion that incorporation
of unnatural amino acids at the 11 sites chosen appears not to
negatively affect the structural integrity of these FAS-TE
mutants.
NMR Spectra of OMePhe Mutant Proteins. 1H-13C and
1H-15N HSQC spectra were recorded for each FAS-TE mutant
labeled with 13C/15N OMePhe (Figure 4). With the exception
of Tyr-2343-OMePhe, a single 1H-13C cross peak was observed
for the methoxy group in a narrow spectral window from 3.55
to 3.82 ppm in the proton and from 54.8 to 56.0 ppm in the
carbon dimension. There was no cross peak observed for Tyr-
2343-OMePhe presumably because of conformational exchange.
Upon addition of the tool compound, a sharp cross peak for
the methoxy peak of Tyr-2343-OMePhe appears at approxi-
mately 2.45 ppm (1H) and 53 ppm (13C). Less dramatic chemical
shift changes are apparent for Leu-2222-OMePhe, Tyr-2347-
OMePhe, Tyr-2351-OMePhe, Gln-2373-OMePhe, Phe-2375-
OMePhe, and Tyr-2454-OMePhe. Interestingly, Tyr-2307-
OMePhe does not show any chemical shift changes despite its
position at the center of the active site, adjacent to the catalytic
residue Ser-2308 (see Figures 7 and 8). This suggests that the
bulky methoxy group inhibits binding. Interestingly, for some
of the proteins (Leu-2222-OMePhe, Gln-2373-OMePhe, Phe-
2375-OMePhe and Tyr-2454-OMePhe) as much as 20% cannot
be converted to a complex even in the presence of a 4-fold molar
excess of the tool compound (data not shown). This is possibly
the result of limited solubility of the tool compound.
Figure 7. Site-directed labeling of active site residue Tyr-2307. After UV
cleavage, 15N-oNBTyr incorporated at Tyr-2307 does, in contrast to OMePhe
and OCF3Phe, not inhibit tool compound binding. Methyl region of 1H NMR
spectra of Tyr-2307-oNBTyr before (A) and after UV cleavage (B), of wild-
type protein before (C) and after addition of tool compound (D), of Tyr-
2307-oNBTyr after UV cleavage and addition of tool compound (E), and
of Tyr-2307-OMePhe before (F) and after addition of tool compound (G).
The position of methyl resonances characteristic of the complex are indicated
by dashed lines.
in large proteins than backbone amide resonances.45,46 This
seems to be the case for the OMePhe mutants of FAS-TE as
well.
NMR Spectra of OCF3Phe Mutant Proteins. 1D 19F-NMR
spectra were recorded for each OCF3Phe FAS-TE mutant
(Figure 5). In each case, a single peak was observed within 0.9
ppm of a resonance line recorded for a 0.5 mM solution of the
unnatural amino acid OCF3Phe, dissolved in the same buffer
and identical conditions. The width of the fluorine resonance
line varied for each position again suggesting conformational
exchange. The chemical shift of fluorine resonances is highly
sensitive to changes in the environment,5–7 and the line widths
are a sensitive monitor of conformational exchange and reflect
conformational fluctuations in the protein.6,7,47
Addition of tool compound results in significant chemical shift
changes for residues near the binding site. In some cases, for
example, for Tyr-2343-OCF3Phe, Tyr-2351-OCF3Phe, His-2408-
OCF3Phe, and Tyr-2454-OCF3Phe compound addition results
in sharpening of the fluorine line; for Gln-2373-OCF3Phe and
Phe-2375-OCF3Phe the opposite is observed. As with Tyr-2307-
The observations for Tyr-2343-OMePhe suggest that parts
of FAS-TE undergo conformational exchange on the millisecond
time scale. Tool compound binding apparently reduces confor-
mational exchange but only partially. 1H-15N HSQC measure-
ments of the same samples support this interpretation as an
amide resonance peak was observed for only six of the 11
mutants even in the presence of 4-fold excess of the tool
compound (Figure S9, Supporting Information). 1H-15N cross
peaks were observed for Leu-2222-OMePhe, Thr-2255-OMe-
Phe, Tyr-2347-OMePhe, His-2408-OMePhe, Thr-2450-OMe-
Phe, and Tyr-2454-OMePhe; with the exception of Leu-2222
and Tyr-2347, these are mutants that show no or only small
chemical shift changes upon compound binding in the 1H-13C
HSQC data (Figure 4). Because of their superior relaxation
behavior, methyl groups are generally more readily observable
(45) Kay, L. E.; Bull, T. E.; Nicholson, L. K.; Griesinger, C.; Schwalbe,
H.; Bax, A.; Torchia, D. A. J. Magn. Reson. 1992, 100, 538–558.
(46) Kay, L. E.; Bull, T. E.; Nicholson, L. K.; Griesinger, C.; Schwalbe,
H.; Bax, A.; Torchia, D. A. J. Magn. Reson. Series A 1994, 111, 231–
232.
(47) Bai, P.; Luo, L.; Peng, Z. Y. Biochemistry 2000, 39, 372–380.
9
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A R T I C L E S
Cellitti et al.
Figure 8. Chemical shift mapping of tool compound 17 binding to FAS-TE. (A) Combined chemical shift data. Chemicals shift changes ∆CS of 19F
resonances (OCF3Phe; purple) were scaled relative to 1H by the gyromagnetic ratio while 1H-13C (OMePhe; violet) and 1H-15N (oNBTyr; pale yellow)
chemical shifts were combined using the expression ∆CS ) [(∆1H)2 + (0.252*∆13C)2]0.5 and ∆CS ) [(∆1H)2 + (0.102*∆15N)2]0.5, respectively. OMePhe
and OCF3Phe at Tyr-2307 block binding (see text). For oNBTyr mutants only a limited set of chemical shift changes could be compiled because of conformational
exchange (see text). (B) Structure of the covalent orlistat complex (2PX6.pdb).30 The average chemical shift changes induced by the binding of 17 are
calculated for each unnatural amino acid and color-coded for each position: ∆CS < 0.1 ppm, blue; 0.1-0.2 ppm, salmon; > 0.2 ppm, red. Disordered loops
are indicated by dashed lines. The active site residues Ser-2308, Asp-2338, and His-2481 are shown in magenta.
OMePhe, no changes are observed for Tyr-2307-OCF3Phe
suggesting again that the addition of the bulky OCF3 (or OMe)
group to this near active site residue blocks binding. The small
chemical shift changes to Thr-2450-OCF3Phe and Thr-2255-
OCF3Phe may be a solvent effect as the tool compound is added
as DMSO stock (<0.5% final DMSO concentration).
the individually 15N-oNBTyr labeled mutant proteins allows the
assignment of five of the tyrosine amide resonances.
As shown above, the data for OMePhe and OCF3Phe indicate
that unnatural amino acid incorporation at Tyr-2307, next to
the active site residue Ser-2308, inhibits binding of the tool
compound presumably because of steric interference by the
added OCH3 or OCF3 group. UV photocleavage of Tyr-2307-
oNBTyr re-establishes binding (Figure 6A) because the natural
binding surface is regenerated as can be clearly seen from the
comparison of 1D 1H spectra of wild-type and mutant proteins
(Figure 7).
1
NMR Spectra of oNBTyr Mutant Proteins. H-15N HSQC
spectra were recorded for each FAS-TE mutant labeled with
15N-oNBTyr (Figure 6) before and after photocleavage by UV
light and after addition of tool compound. As expected from
1
the H-15N HSQC data of OMePhe mutants (Supplemental
Figure S9), cross peaks are observed for only a subset of
mutants, namely Leu-2222-oNBTyr, Tyr-2351-oNBTyr, Gln-
2373-oNBTyr, Phe-2375-oNBTyr, His-2408-oNBTyr, Thr-2450-
oNBTyr, and Tyr-2454-oNBTyr. UV cleavage of the photo-
caging group results in modest chemical shift changes for four
of these mutants while the signal for the three others disappears
presumably because of conformational exchange (Figure 6). For
the five mutants that are tyrosine in wild-type FAS-TE, only
Tyr-2307 is observed in the UV-cleaved, uncomplexed form.
Its chemical shift corresponds to an observable cross peak in
the spectrum of wild-type apoprotein selectively labeled with
15N-tyrosine (Figure 6B). In the latter spectrum, only nine of
the 16 expected cross peaks are observed indicating that seven
tyrosines undergo conformational exchange or rapid exchange
with bulk solvent. After the addition of tool compound 15 of
the 16 tyrosines (Figure 4D) are observed with various intensities
and line widths suggesting that line broadening and absence of
signals in the uncomplexed protein is indeed caused by
conformational exchange (the last tyrosine is part of the
presumably unstructured expression tag). The comparison with
Mapping the Binding Site of a Tool Compound. The tool
compound binds tightly to all FAS-TE mutants (except for Tyr-
2307-OMePhe and Tyr-2307-OCF3Phe). Binding occurs in the
1
slow exchange binding regime for all H, 13C, 15N, and 19F
resonances. The chemical shift changes induced by compound
addition map the binding site of the ligand to the active site of
FAS-TE (Figure 8). Data obtained from 1H-13C, 1H-15N and
19F NMR spectra are all consistent in that His-2408, Thr-2450
and Thr-2255 mutants are not affected by binding. The most
pronounced chemical shift changes occur at residue Tyr-2343
that is apparently disordered in the uncomplexed proteins (by
NMR but not in the crystal structures of apo FAS-TE32 and of
the orlistat complex30). Increasingly smaller chemical shift
changes are observed for neighboring Tyr-2347 and Tyr-2351
mutants. Compound binding most likely stabilizes this flexible
region of the protein as suggested by sharper fluorine resonance
lines at residues Tyr-2343, Tyr-2351, and Tyr-2454 (Figure 5).
Modest chemical shift changes are also observed for Gln-2373
and Phe-2375 mutants at the top surface of the binding pocket.
At the far right site of the pocket, among the largest chemical
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In vivo Incorporation of Unnatural Amino Acids
A R T I C L E S
shift changes are observed for Leu-2222 mutants whereas Thr-
2255 and His-2408 mutant resonances are not shifted by
compound binding. On the other end, unnatural amino acids at
residue Thr-2450 and Tyr-2454 are also not affected, observa-
tions that are consistent with the overall features of the orlistat
complex.30
served probably because they contact the ring and not the
para substitution. As in other evolved tRNA synthetases,
residues Tyr32 and Asp158 are mutated to remove H-bonds
to the hydroxyl group of natural tyrosine. In the OMePhe
RS, the mutations Tyr32Gln, Asp158Ala, and Leu162Pro
increase the size of the binding pocket to accommodate the
additional methyl group. Comparing the newly evolved
OCF3Phe RS to the OMePhe RS suggests that the OCF3 group
requires more space in the active site, as the OCF3Phe RS
repeats the Asp158Ala mutation but also has other mutations
(Tyr32Ala, Leu65Ser) that further reduce the bulk of side
chains near the OCF3 group (based on the structure of the
OMePhe RS).49 Interestingly position 109 is mutated from a
hydrophilic Gln to a hydrophobic residue of variable size,
possibly because of the suggested higher hydrophobicity of
the OCF3 group (relative to OCH3). Finally, residues 108
and 162, which point outward in the OMePhe structure, seem
to be less important in the case of OCF3Phe, as they are
mutated to a wide variety of side chains in the selection
winners.
The fidelity of amino acid incorporation of the newly
evolved RS used in conjunction with the OMePhe tRNA13
was evaluated by mass spectrometric methods38 and found
to be better than 99.5%. This suggests that it should be
possible to evolve specific tRNA synthetases for other
fluorinated, especially trifluoro-substituted amino acids. These
unnatural amino acids do not have to be enantiomerically
pure. Racemic mixtures of OCF3Phe (this study) and of
several other unnatural amino acids acids50 have been used
successfully to evolve selective RS mutants that retain their
specificity for L-amino acids presumably because the residues
randomized in the selection library are surrounding the end
of the amino acid side chain rather than participating in
backbone recognition. For FAS-TE, selective incorporation
of L-OCF3Phe is suggested by the unperturbed 1H NMR
spectra although DL-OCF3Phe was used in the incorporation
experiment (Figure S6, Supporting Information, see below).
Incorporation of Three Unnatural Amino Acids at 11
Different Sites. Our results indicate that all three unnatural amino
acids (supplied as L-amino acids for OMePhe and oNBTyr and
as DL-racemic mixture for OCF3Phe) can be incorporated at
all 11 sites in a 33 kDa protein with good yields (Table 1) and
with little effect on the structure of the mutant proteins (Figure
3, Figures S4-S8, Supporting Information). As with single-
site mutations using natural amino acids, unnatural amino acid
incorporations can interfere with protein structure and stability.
In the current study, care was taken to introduce the unnatural
amino acids at solvent exposed sites. Subsequently, with the
exception of incorporation at Tyr-2307 adjacent to the active
site, no effect on the ability of FAS-TE mutants to bind a tool
compound in the active site was observed (see below). However,
the observed variations in expression yields may reflect desta-
bilizing effects on some of the mutations or context dependent
variations in incorporation efficiencies, and warrant future
investigations.
Discussion
Improving Incorporation Efficiency. NMR studies require
milligram quantities of isotopically labeled protein. In our
previous proof-of-concept experiment, we incorporated 15N-
labeled OMePhe using a mutant M. jannaschii tyrosyl tRNA/
tRNA synthetase pair into myoglobin.19 Approximately 0.5 mg
of protein was produced from 0.5 L of E. coli culture in defined
minimal media (GMML) using ∼100 mg of the custom
synthesized unnatural amino acid to yield a single 55 µM NMR
sample.
Initial incorporation tests in rich rather than defined minimal
media with a well-expressing FAS-TE construct indicated that
most of the protein produced was truncated at the TAG
incorporation codon (Figure S1, Supporting Information). This
is in contrast to findings in GMML media where yields of full-
length mutant protein can reach up to 70% of that of wild-type
for some proteins.34 At the higher expression levels achieved
in rich media the amount of amber tRNA aminoacylated with
the unnatural amino acid is apparently insufficient. To overcome
this limitation, we modified the incorporation plasmid and added
a copy of the orthogonal RS under the control of an inducible
promoter (Figure 2). Simultaneous overexpression of the target
protein and of the RS dramatically reduced truncation at the
TAG incorporation codon (Figure S1, Supporting Information).
This modification of the plasmid appears to be beneficial for
the incorporation of at least three different unnatural amino
acids: OMePhe, OCF3Phe, and oNBTyr. Using standard shake
flask cultures and rich media, single NMR samples from 50
mL of E. coli culture were obtained for most mutants using
only 8 mg of oNBTyr, 12.5 mg of OCF3Phe, or 25 mg of
OMePhe (see details below). For well-expressing protein such
as FAS-TE, the current system allows for the economical
production of protein NMR samples in rich growth media and
as such should be widely applicable.
Selecting a New tRNA Synthetase for a Fluorinated Un-
natural Amino Acid. As discussed above, fluorinated unnatural
amino acids and OCF3Phe in particular represent attractive NMR
labels. Consequently an expression system for incorporating
OCF3Phe into proteins was established. Initially, we attempted
to express proteins with OCF3Phe using the existing OMePhe
RS and its amber tRNA,13 but the two amino acids are
sufficiently different that this RS does not facilitate incorporation
of OCF3Phe into proteins. In hindsight, this is not surprising as
a CF3 group should be ∼70% bulkier than CH3 (C-F bond
length 1.39 Å vs C-H 1.09 Å; van der Waals radii of 1.35 Å
for F vs 1.2 Å for H), and may be more hydrophobic as
suggested by partitioning studies of hexafluoroleucine and
leucine48 (also see ref 42 and references therein).
Monitoring Conformational Exchange. At 33 kDa, FAS-TE
is a sizable protein and normally deuterium labeling is
employed in order to minimize spin diffusion and increase
sensitivity and resolution.51 With the exception of Tyr-2343-
Using three libraries that randomize positions Tyr32,
Leu65, Asp158, Leu162, and either His70 and Gln155 (L1)
or Phe108 and Gln109 (L2)34 or His70, Phe108, Gln109,
Gln155, and Ile159 (L3D)35 several tRNA synthetases specific
for OCF3Phe were selected. His70 and Gln155 were con-
(49) Zhang, Y.; Wang, L.; Schultz, P. G.; Wilson, I. A. Protein Sci. 2005,
14, 1340–1349.
(48) Lee, K. H.; Lee, H. Y.; Slutsky, M. M.; Anderson, J. T.; Marsh, E. N.
(50) Santoro, S. W.; Wang, L.; Herberich, B.; King, D. S.; Schultz, P. G.
Nat. Biotechnol. 2002, 20, 1044–1048.
Biochemistry 2004, 43, 16277–16284.
9
J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008 9279

A R T I C L E S
Cellitti et al.
OMePhe, all of the methoxy groups of the 11 OMePhe
mutants are observed (Figure 4). Solvent exposed methoxy
groups may be less sensitive to exchanges processes because
of smaller chemical shift differences between the exchanging
forms, and may be more readily observed because of the
generally favorable relaxation properties of methyl groups.
orlistat in its hydrolyzed form and with both hexanoyl and
the beginning of the palmitic core in the covalent orlistat
complex (Figure 8). Tyr-2343, together with Tyr-2347 and
Tyr-2351 are part of an R-helix that is observed in only one
of two asymmetric units of an unpublished crystal structure
(G. Spraggon, B. Bursulaya et al., unpublished data) and is
absent in all published structures. Based on the sharper
resonance lines, it is possible that tool compound binding
stabilizes this helix but a more detail analysis must await a
cocrystal structure or more detailed NMR studies. Because
of the inherent flexibility of this part of the binding pocket
we have not attempted to model binding of the tool compound
but all preliminary evidence suggests a compound localization
similar to that of orlistat.
1
The small H-1H dipole-dipole couplings of the methoxy
protons to the ring protons may be beneficial as well. For
Tyr-2343-OMePhe conformational exchange most likely
causes line broadening as addition of the tool compound gives
rise to a significantly shifted resonance line. This interpreta-
tion is supported by 1H-15N data obtained with 13C/15N-
OMePhe mutants (Figure S9, Supporting Information), 15N-
oNBTyr labeled mutant proteins and wild-type protein
selectively labeled with 15N-tyrosine (Figures 6 and S8,
Supporting Information): Before addition of the tool com-
pound amide signals of seven tyrosines including those of
Tyr-2343, Tyr-2347, Tyr-2351 and Tyr-2454 are exchange
broadened. Additional evidence for conformational exchange
is also provided by 19F NMR data with OCF3Phe mutants.
19F chemical shifts of fluorinated analogs of natural amino
acids have long been known to be very sensitive to side chain
dynamics.6,7,47 Line broadening is evident for incorporation
at the same FAS-TE location for different unnatural amino
acids. Resonance lines that are most affected correspond to
residues that have high B-factors or missing electron densities
in the available crystal structures (residues 2325 to 2330,
2344 to 2356, 2452 to 2457, and 2487 are missing in the
published X-ray structure of apo-FAS-TE, 1XKT.pdb,32 and
similarly in the orlistat complex, 2PX6.pdb30), suggesting
conformational heterogeneity for these regions of the protein.
This suggests that the NMR observations are indeed the result
of conformational exchange processes in the natural protein
and not the result of the unnatural amino acid incorporation
into mutant FAS-TE. These observations in turn indicate that
unnatural amino acids can be used to probe protein structure
and dynamics at specific sites in large proteins.
Chemical Shift Mapping of a Small Molecule Binding Site
in FAS-TE. Although different in absolute value, trends and
magnitude of the chemical shift changes induced by tool
compound binding in OMePhe, OCF3Phe and oNBTyr
mutants agree and identify the active site of FAS-TE as the
binding pocket (Figure 8). Data obtained from 1H-13C,
1H-15N and 19F NMR spectra are all consistent in that
residues His-2408, Thr-2450 and Thr-2255 mutants are not
affected by binding. When compared to the crystal structure
of the FAS-TE complex with orlistat,30 Thr-2255 and His-
2408 are at least 12 Å from the closest orlistat atom while
the distance to Thr-2450 is 16 Å. Similarly, mutants of Thr-
2450 exhibits minimal chemical shift changes as this residue
is more than 13 Å from any orlistat atom. These observations
indicate that the tool compound must bind in the active site
not unlike orlistat. The similarities between the orlistat
complexes and the complex of the tool compound are further
supported by large chemical shift changes for Leu-2222
mutants. Leu-2222, therefore likely marks the extent of the
compound binding pocket on the right side. The residue with
the most pronounced chemical shift changes for its mutants,
Tyr-2343, forms close contacts with the hexanoyl tail of
The initial observations for Tyr-2307 mutants are mislead-
ing because introducing a bulky CH3 or CF3 group at the
end of the unnatural amino acids instead of the natural
tyrosine side chain apparently interferes with binding. UV
cleavage of oNBTyr incorporated at Tyr-2307 restores
binding (Figure 7) suggesting that the binding surface was
perturbed in the Tyr-2307-OMePhe and Tyr-2307-OCF3Phe
mutant proteins. As with any type of mutation there is the
need to carefully consider the sites of change because altering
critical residues to unnatural amino acids may impact protein
function. Clearly photocaged unnatural amino acids constitute
an attractive and unique labeling strategy as, with for example
oNBTyr, a single NMR label can be introduced at a selected
tyrosine residue without perturbing the primary protein
sequence.
Overall, the current study illustrates the robustness of our
improved in ViVo incorporation protocol. The three unnatural
NMR-active amino acids can now potentially be employed
to applications ranging from screening for site-selective
binders to mapping small-molecule target interactions in the
absence of a crystal structure. Most interesting will be
applications with very large proteins with thousands of
residues for which obtaining signal assignments by traditional
approaches is a truly formidable challenge. A recent test
incorporation of NMR-acitve unnatural amino acids into fully
deuterated FAS-TE is a first step toward such applications.
Conclusions
Three different NMR-active unnatural amino acids were
incorporated with high efficiency at different sites in a 33 kDa
protein of pharmaceutical interest without adversely effecting
protein structure and ligand binding. As expected, 13C-labeled
methyl groups introduced with the unnatural amino acid, because
of their relaxation behavior, proved to be particularly advanta-
geous for studies of a large protein. As illustrated here, unnatural
amino acid labeling can be used to probe conformational
exchange processes and small molecule ligand binding. More
specifically, tool compound binding induces conformational
rearrangements near the active site of FAS-TE and stabilizes
part of the binding surface that is flexible in the X-ray structure
of the orlistat complex. Although all mutations were well
tolerated, introducing OMePhe or OCF3Phe at one active site
residue inhibited binding of a tool compound. The same site
was probed with a photocaged oNBTyr that offers the advantage
of introducing NMR labels at specific tyrosine residues without
altering the protein sequence and therefore binding surface.
Together with oNBCys (ref 52 and M. Jahnz, D. Summerer,
B.H. Geierstanger, P.G. Schultz, unpublished results) and
(51) Gardner, K. H.; Kay, L. E. Annu. ReV. Biophys. Biomol. Struct. 1998,
27, 357–406.
(52) Wu, N.; Deiters, A.; Cropp, T. A.; King, D.; Schultz, P. G. J. Am.
Chem. Soc. 2004, 126, 14306–14307.
9
9280 J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008

In vivo Incorporation of Unnatural Amino Acids
A R T I C L E S
o-nitroveratryl-Ser,53 the concept of photocaged unnatural amino
acid incorporation opens new and exciting avenues for site-
specific labeling and should therefore enable NMR studies of
large proteins that can currently not be studies by traditional
methods.
helpful discussions and comments. This work was in part supported
by grants from the National Institutes of Health (GM62159 to
P.G.S.).
Supporting Information Available: Sequence information of
all newly selected tRNA synthetases, SDS gels of incorporation
tests, gel and MS data of OCF3Phe incorporation, and additional
NMR data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment. We thank Jianqiao Lin for help with protein
expression and purification for some of the oNBTyr mutants,
Genevieve Welch for help with Figure 2, and Dr. Scott Lesley for
(53) Lemke, E. A.; Summerer, D.; Geierstanger, B. H.; Brittain, S. M.;
Schultz, P. G. Nat. Chem. Biol. 2007, 3, 769–772.
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