Efficient synthesis and in vivo incorporation of acridon-2-ylalanine, a fluorescent amino acid for lifetime and Foerster resonance energy transfer/luminescence resonance energy transfer studies

Article abstract of DOI:10.1021/ja403247j

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in Escherichia coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here, we report the synthesis of Acd in 87% yield over five steps from Tyr and the identification of an Acd synthetase by screening candidate enzymes previously evolved from Methanococcus janaschii Tyr synthetase for unnatural amino acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with select natural amino acids and Foerster resonance energy transfer (FRET) interactions with common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu³⁺ to monitor protein folding and binding interactions.

Full text of DOI:10.1021/ja403247j

Article
pubs.acs.org/JACS
Efficient Synthesis and In Vivo Incorporation of Acridon-2-ylalanine,
a Fluorescent Amino Acid for Lifetime and Forster Resonance Energy
̈
Transfer/Luminescence Resonance Energy Transfer Studies
†
†
†
†
†
Lee C. Speight, Anand K. Muthusamy, Jacob M. Goldberg, John B. Warner, Rebecca F. Wissner,
‡
‡
,‡
,†
Taylor S. Willi, Bradley F. Woodman, Ryan A. Mehl,* and E. James Petersson*
†University of Pennsylvania, Department of Chemistry, 231 South 34th Street, Philadelphia, Pennsylvania, 19104-6323, United States
‡Oregon State University, Department of Biochemistry and Biophysics, 2011 Ag Life Sciences Building, Corvallis, Oregon
97331-7305, United States
*
S Supporting Information
ABSTRACT: The amino acid acridon-2-ylalanine (Acd) can
be a valuable probe of protein conformational change because
it is a long lifetime, visible wavelength fluorophore that is small
enough to be incorporated during ribosomal biosynthesis.
Incorporation of Acd into proteins expressed in Escherichia coli
requires efficient chemical synthesis to produce large quantities
of the amino acid and the generation of a mutant aminoacyl
tRNA synthetase that can selectively charge the amino acid
onto a tRNA. Here, we report the synthesis of Acd in 87%
yield over five steps from Tyr and the identification of an Acd
synthetase by screening candidate enzymes previously evolved from Methanococcus janaschii Tyr synthetase for unnatural amino
acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with
̈
select natural amino acids and Forster resonance energy transfer (FRET) interactions with common fluorophores such as
methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd
fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu3+ to monitor protein folding and binding interactions.
INTRODUCTION
(GFP) or its derivatives, but the large size of GFP may perturb
■
6
−8
the folding event of interest.
Smaller synthetic fluorophores
Fluorescence spectroscopy is a powerful technique for
monitoring protein folding and dynamics. Fluorescence experi-
ments can be carried out with nanosecond time resolution, at
the single molecule level, and in real time in living cells.
Structural information can be derived from fluorescence
experiments by exploiting distance-dependent interactions
can often be attached in an amino acid specific manner, fo9r
example, by exploiting the unusual reactivity of the Cys thiol.
However, if one wishes to carry out experiments in vivo or in
cell lysate, reaction with a specific Cys is extremely difficult to
achieve.
To gain greater specificity in vivo, tag sequences can be
inserted in the protein, which are then modified. These tag
sequences range from sizes comparable to GFP to stretches of
such as Fo
̈
rster resonance energy transfer (FRET) or
fluorescence quenching through photoinduced electron transfer
1
−5
(
PET).
Making such measurements in a time-resolved
1
0,11
only six amino acids.
The tag modification can be purely
manner allows for the tracking of protein conformation as two
regions of the protein undergo relative translational and
rotational motion. The developments in spectroscopy over
the last 25 years have afforded tremendous improvements in
sensitivity as well as temporal and spatial resolution. Today, the
information that one can derive from these experiments is often
limited not by instrumentation but by one’s ability to label the
protein in an efficient, site-specific, and nonperturbing manner.
Many strategies exist for fluorescently labeling proteins, each
with advantages and drawbacks. Use of the intrinsic
fluorescence of Trp and Tyr is often limited to relatively
small or simple proteins because most large proteins have
multiple Trp or Tyr residues, complicating the interpretation of
fluorescence data. The most common extrinsic labeling
strategies use genetic fusions of green fluorescent protein
chemical such as the binding of arsenical fluorophore
compounds (e.g., FlAsH) to a CCPGCC motif, or it can be a
12−23
more complex chemoenzymatic process.
While these are
valuable methods for labeling proteins, fluorescent amino acids
that are incorporated cotranslationally have the potential to
provide much more information on protein folding. They can
be used to monitor proteins as they fold on the ribosome, and
they can be placed at interior locations that are unavailable to
24
modification after folding of the protein. Several examples of
incorporation of fluorescent amino acids have been demon-
strated by semisynthesis of aminoacyl tRNA that is then used
Received: April 1, 2013
©
XXXX American Chemical Society
A
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for in vitro translation with reconstituted ribosomes or injection
25−29
into Xenopus oocytes.
To our knowledge, only one visible-
wavelength fluorescent amino acid has been shown to be
compatible with site-specific in vivo incorporation in Escherichia
30−36
coli (also two in eukaryotic cells).
Here, we develop methods for the efficient synthesis and in
vivo incorporation of acridon-2-ylalanine (1, Acd, δ), a blue-
wavelength fluorescent amino acid. Acd is a useful fluorophore
Figure 1. Unnatural amino acids used in this study. Aminoacyl tRNA
synthetases (RSs) were screened for successful incorporation of
acridon-2-ylalanine (1, Acd, δ) from an RS pool previously selected for
incorporation of 4-(2′-bromoisobutyrlamido)phenylalanine (2, Brb, β)
and p-benzoylphenylalanine (3, Bzf, ζ).
3
because of its small size (222 Å ), near unity quantum yield in
water (Φ = 0.95), unusually long lifetime (τ ∼ 15 ns) and high
37−39
photostability (<5% degradation after 3 h irradiation).
Previous work in the Petersson laboratory has shown that Acd
can be efficiently quenched by a thioamide through a PET
3
7
mechanism. This makes the Acd/thioamide pair an extremely
small, nonperturbing set of labels since Acd is only 47% larger
than Trp, and the thioamide is essentially isosteric with an
amide. In addition, Acd can also act as a FRET partner with
appropriately matched fluorophores, and Acd can be used to
sensitize Eu3 emission via a luminescence resonance energy
Tyr analogues; therefore, we suspected that these UaaRSs could
48,49
accept Acd.
Indeed, we identified a useful synthetase from
this library and characterized its efficiency of incorporation of
Acd into model proteins. We also analyzed a series of mutants
containing some active site residues from these selected
UaaRSs and found that selectivity for Acd over Tyr depends
upon a few key mutations.
+
4
0,41
transfer (LRET) mechanism.
Sisido and co-workers have previously shown that Acd can be
ribosomally incorporated into proteins by in vitro translation
In order to provide other investigators with important
photophysical parameters for the use of Acd, we have
characterized its quenching by several natural amino acids
38
using the PURE system. However, protein yields from
systems using semisynthetic tRNA are inherently limited
because the aminoacyl tRNA is rapidly hydrolyzed in the
translation mixture. Also, in vitro translation on the scale
necessary to produce milligrams of protein for biochemical
studies is not often practiced and may require specialized
̈
through Stern−Volmer titrations and determined the Forster
radius, or distance of half-maximal energy transfer (R ), for
0
FRET to common fluorophores such as BODIPY Fl and
fluorescein. Finally, we expressed several proteins to demon-
strate the usage of Acd fluorescence in monitoring conforma-
tional changes and protein binding to peptide or ions through
steady state or fluorescence lifetime measurements.
4
2,43
expertise.
On the other hand, the in vivo incorporation
methods pioneered by Schultz and co-workers are much more
4
4,45
broadly enabling to the biochemical community.
These
methods require the generation of an aminoacyl tRNA
synthetase (RS) that is selective for an unnatural amino acid
RESULTS AND DISCUSSION
■
Although we relied on a previously published synthetic route
for our earlier experiments, when we began to contemplate in
vivo incorporation, the large quantities of Acd required for
expression in bacterial culture led us to consider a more cost-
effective route. Acd syntheses reported by Lankiewicz and
(Uaa). The UaaRS charges a cognate tRNA that recognizes the
UAG stop codon (tRNACUA). The UAG stop codon is inserted
into the gene for the protein target at the site of interest, while
a different stop codon (e.g., UGA) is used for translation
termination. After aminoacylation by the UaaRS, the tRNACUA
delivers the unnatural amino acid to the site of interest in the
protein during ribosomal biosynthesis. Plasmids coding for the
UaaRS, tRNACUA, and protein of interest are transformed into
E. coli. After growth in the presence of the Uaa, protein
containing the Uaa is purified for use in biochemical
experiments. Because tRNACUA must compete with release
factors, protein production levels are lower than those for
normal expression, but one can generally obtain 1−10 mg/L
38,39
Sisido used p-nitrophenylalanine as the starting material.
After hydrogenation, N-Boc-p-aminophenylalanine ester was
coupled to 2-iodobenzoic acid in an Ullman amine synthesis to
give a product like 6. Acid-catalyzed Friedel−Crafts cyclization
in polyphosphoric acid (PPA) gave Acd (1). Further protecting
group manipulations were used to obtain Acd in an appropriate
form for peptide coupling or in vitro aminoacylation of
38,39
tRNA.
We wished to improve the synthesis by starting from
the natural amino acid Tyr and using a Buchwald−Hartwig
46
quantities of protein from cultures.
coupling to the commodity chemical O-methyl anthranilate (5)
50
A typical protein expression requires millimolar concen-
trations of the unnatural amino acid in the media. Thus, a
highly efficient synthesis is required to produce the gram
quantities of amino acid necessary for routine liter scale protein
expression. Therefore, we began by designing a new synthesis
of Acd starting from Tyr that is efficient in terms of labor and
materials costs. This route also has the advantage of versatility,
as substituted Acd derivatives can easily be prepared from Tyr
analogs.
to form 6. We also found heating in sulfuric acid with the
addition of water to be a preferable way to induce Friedel−
Crafts cyclization, as it resulted in deprotection of the Boc
group and methyl esters as well the formation of Acd. Since
preparation of 4 is straightforward, the efficiency of the route in
Scheme 1 relied on developing a high yielding cross-coupling
reaction.
In order to identify optimal conditions for the Pd-catalyzed
coupling of 4 and 5, we screened a number of combinations of
solvent, temperature, base, and Pd/ligand ratios. From these
experiments, we determined that the highest yields were
To obtain a UaaRS for incorporating Acd into proteins
expressed in E. coli, we screened UaaRSs previously selected for
incorporation of the structurally similar amino acids 4-(2′-
bromoisobutyramido)phenylalanine (2, Brb, β) and p-benzoyl-
obtained in 4 h at 120 °C with 5.2 mol % Pd(OAc) , 3.75 mol
2
% racemic 2,2′-bis(diphenyl-phosphino)-1,1′-binaphthyl (rac-
47
phenylalanine (3, Bzf, ζ) shown in Figure 1. Similar UaaRSs
derived from Methanococcus janaschii TyrRS had been shown
by the Mehl laboratory to be permissive of a variety of bulky
BINAP), and 3.0 equiv Cs CO as base in cyclopentyl methyl
2
3
ether (CPME). The results of our screen are shown in
Supporting Information.
B
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Scheme 1. Optimized Synthesis of Acridon-2-ylalanine
Figure 2. Acridon-2-ylalanine chromophore interactions. Left:
Quenching by natural amino acids assessed through steady state
fluorescence Stern−Volmer titrations with five amino acids: Trp (filled
red circles), Met (orange squares), Cys (green upright triangles), His
(
pale blue downward triangles), Tyr (filled dark blue diamonds). Tyr
After screening in microplates was done, we carried out
reactions on 250 mg and 1 g of 4 to ensure that our yields
could translate to those scales. We found that 92% yields could
be obtained from the optimal conditions, at a larger scale with
toluene as the solvent (chosen based on cost since results were
comparable to CPME), 135 °C heating, and a 23 h reaction
time. Friedel−Crafts cyclization and global deprotection could
be achieved in 98% yield, giving 87% overall yields of Acd on
the 1 g scale.
methyl ester used for solubility, all other amino acids used as free acid.
Left inset: Fluorescence lifetime Stern−Volmer titrations with Trp
(open red circles) and Tyr (open dark blue diamonds). Steady state
data shown as solid lines for comparison. Right: Absorbance (gray)
and fluorescence emission (light blue) of Mcm and absorbance of Acd
(
blue), with spectral overlap shaded. Absorbance spectra are
normalized by extinction coefficient; the emission spectrum scaling
is arbitrary.
In order to understand how best to use Acd, we began by
assessing its sensitivity to local environment. This is clearly
important if one wishes to use Acd as a probe of protein
environment, but it is also an important background correction
for those who wish to use Acd in PET or FRET studies. Acd
has previously been shown to have an inherent solvatochromic
fluorophores such as carboxyfluorescein (R = 51 Å), BODIPY
0
Fl (R = 49 Å), rhodamine 6G (R = 49 Å), or nitro-
0
0
benzodioxazole (NBD, R = 37 Å). (The spectra used to
0
determine these values are given in Figures S2−S4 in
Supporting Information.) These values were calculated using
a donor quantum yield (Φ ) of 0.95 for Acd. Since, as we have
D
3
9
response. Its spectral shape blue shifts and its quantum yield
changes from 0.95 to 0.29 on moving from water to THF.
However, quenching of fluorophores by specific amino acids
just shown, Acd quantum yield can vary with local environ-
ment, any Acd FRET donor experiment must be compared to
an equivalent experiment with an Acd label alone to correct for
51,52
can also lead to environmental sensitivity.
As noted above,
these effects on Acd Φ .
D
we have recently shown that Acd fluorescence can be quenched
Although Sisido and co-workers had previously shown that
Acd could be incorporated into proteins by in vitro ribosomal
synthesis, we wished to produce milligram quantities of protein
for biophysical studies. Therefore, we sought to obtain a UaaRS
37
by a thioamide through a PET mechanism. To determine the
effects of natural amino acids on Acd fluorescence, we
performed Stern−Volmer titrations with the common redox-
active amino acids: Trp, Tyr, Met, Cys, and His. As seen in
Figure 2, only Trp and Tyr quench Acd significantly.
38
that would allow us to incorporate Acd in vivo. Since Acd is
significantly larger than Tyr, we began not by selecting from
mutant TyrRS but by screening a library of synthetase mutants
already selected for incorporation of Brb, Bzf, or 2-
napthylalanine (Nap). It had previously been shown that for
some amino acids that deviate substantially from the starting
structure, it can be difficult to alter the binding pocket
sufficiently to recognize the new amino acid in a single round of
Comparisons of data from steady state and fluorescence
lifetime measurements indicate that Trp and Tyr quench Acd
through a PET mechanism with both static (i.e., a nonemissive
ground state complex is formed) and dynamic (i.e., excited
state electron transfer) components. This can be seen in the
Figure 2 inset where the dotted lines show the dynamic
component observed in lifetime measurements made by time-
30
mutagenesis. We also were intrigued by the idea that since
selection protocols only include negative selection against
natural amino acids, mutations that “open up” the binding
pocket could lead to synthetases that are generally permissive of
bulky unnatural amino acids, yet still selective against all of the
53
correlated single photon counting (TCSPC). Since Acd is
quenched by Trp and Tyr, it should be sensitive to protein
motions that change the proximity of these residues.
Acd can also function as a FRET acceptor for Trp because
the tail of the Trp emission spectrum overlaps significantly with
Acd absorbance (see Figure S2, Supporting Information). The
Forster radius for this FRET pair is 23 Å, making it useful in
̈
monitoring distance changes in the 13−37 Å range. Since Trp
can also act as a quencher of Acd through PET, care must be
taken in interpreting FRET measurements at short distances
4
8,49,54,55
natural amino acids.
Since these UaaRSs would not be
challenged by multiple unnatural amino acids simultaneously, in
theory, they would have perfectly adequate fidelity for protein
production.
Screening was carried out by directing incorporation to a
TAG site at position 150 in GFP and assessing the fluorescence
of suspended cells (see Figure 3). Measured fluorescence
reports on the production of full-length GFP, which indicates
the ability of the synthetase to charge tRNACUA with some
amino acid, either Acd or a natural amino acid. Comparison of
GFP production in cultures with Acd to cultures with only the
(
<15 Å) where both processes may be operative. To form a
selectively excitable FRET pair, Acd can act as an acceptor for
-methoxycoumarinylalanine (Mcm). For the Mcm/Acd pair,
7
R is 25 Å, making them useful in the 15−45 Å range. Acd can
0
also function as a FRET donor with many common organic
C
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Journal of the American Chemical Society
Article
Table S1). In general, we obtained yields of purified protein
that ranged from as low as 3% to as high as 69% of the yields of
an equivalent expression of the wild type (WT) protein. While
this variation may seem large, it is consistent with reports of
changes in stop codon suppression efficiency with various
4
2,43,57,58
proteins and positions.
In order to demonstrate the utility of Acd as a fluorescent
2+
probe, we made several mutants of the Ca -binding protein
CaM, placing Acd at sites that were both buried and surface
exposed (F , G , Y100, and L113). We chose CaM because it
13
41
has been structurally characterized with a variety of substrates
bound, and it has been extensively studied in biophysical
experiments. All mutants were expressed in E. coli and purified
by phenyl sepharose chromatography, and then their folding
was assessed by circular dichroism (CD) spectroscopy with and
Figure 3. Synthetase selection and confirmation of Acd incorporation.
Protein expression levels determined from fluorescence of GFP
reporter construct containing a TAG stop codon at position 150.
Fluorescence from full-length GFP indicates successful incorporation
of an amino acid (Acd or Bzf) from a charged tRNACUA. Comparison
to expressions containing only proteinogenic amino acids indicates
UaaRS fidelity (Neg). Fluorescence from WT GFP (black) is shown as
a positive control.
2+
without Ca . The melting points (Tms), determined by
2+
thermal scans in the absence of Ca , were within 5 °C of WT
CaM (see Figure S16, Supporting Information). None of the
2+
mutants denatured in the presence of Ca .
natural amino acids reports on the selectivity of the synthetase
against Tyr. Although none of the BzfRSs or NapRSs showed
evidence of noticeable Acd incorporation, we found that
BrbRS1 and BrbRS3 could efficiently incorporate Acd while
excluding Tyr. It is interesting that the NapRS was able to
charge tRNACUA with Bzf but not Acd. This may be attributable
to the absence of Glu in the NapRS, which may interact with
6
5
the acridone N−H. The active site sequences of all screened RS
mutants are given in Supporting Information. Since BrbRS1
showed higher levels of protein expression, we used it for all
subsequent Acd incorporation and refer to it as AcdRS.
We were curious to understand how AcdRS could be
permissive of Brb and Acd, yet selective against Tyr. AcdRS
contains five mutations relative to the parent M. janaschii
TyrRS. We found that a single mutation of Glu Val was
Figure 4. Calmodulin (CaM) labeling sites. An image showing Acd
(blue) at position 13, 41, 100, or 113 in CaM (red) and highlighting
position 1 in the pOCNC peptide (green). Ca2 ions are shown as
+
6
5
purple spheres. Adapted from PDB 1SYD using PyMol (Schro
̈
dinger,
sufficient to remove its ability to exclude Tyr (see Figure S8,
59
LLC; New York, NY) and Spartan (Wavefunction, Inc.; Irvine, CA).
Supporting Information). Similar results have been observed by
56
Tipmann for other UaaRSs. This is probably generally true of
TyrRS variants evolved for the incorporation of large amino
acids. The pocket must be large enough to accommodate the
Uaa and yet not permit Tyr to bind with hydrating water
molecules. It is perhaps not surprising that such a balance is
easily broken. Additionally, we found evidence of misincorpo-
ration of N-phenyl-p-aminophenylalanine (Npf) resulting from
decarboxylation during the global deprotection/cyclization step
of Acd synthesis (see Figure S13, Supporting Information).
This was true even when Npf was present as only a trace (<1%
by HPLC) impurity in the Acd batch (see Figure S13,
Supporting Information). Since this byproduct, which is
difficult to remove by silica chromatography, is more likely to
be obtained using sulfuric acid, a two-step protocol was adopted
with saponification using LiOH followed by PPA cyclization
We wished to use Acd to monitor conformational changes
associated with the binding of helical peptides to CaM. First, we
used native PAGE gel analysis to confirm that Acd mutation did
not disrupt the binding of our target peptide, pOCNC (H N-
2
FRRIARLVGVLREFAFR-CONH ). This peptide is derived
2
from an olfactory cyclic nucleotide gated ion channel. The
CaM/pOCNC structure has been previously determined by
59
NMR. Its binding affinity to WT CaM is 3 nM; therefore, we
expected that only significant alterations in CaM structure
60
would disrupt binding at 1−10 μM. Indeed, all four Acd
mutants bound a labeled derivative of pOCNC (see Figure S15,
Supporting Information).
We then analyzed the fluorescence emission spectra of 10
μM CaM with varying concentrations of pOCNC. The
fluorescence emission of CaM-F δ, CaM-Y100δ, and CaM-
(54% overall yield). Protein expressed using Acd batches from
13
this PPA route consistently yielded pure protein containing no
Npf at the site of interest (see Figure S14, Supporting
Information). Efforts are currently underway to carry out
negative selections to eliminate Npf misincorporation.
Once this synthetase mutant was identified, we tested
incorporation by expressing Acd mutants of α-synuclein (αS,
see Supporting Information), triose phosphate isomerase
L113δ were unchanged, implying that Acd does not change
environment during the binding of pOCNC (see Figure S18,
Supporting Information). However, in its unbound form, CaM-
G δ showed a blue shift in fluorescence relative to free Acd,
and this blue shift went away as pOCNC was added to the
solution (see Figure S18, Supporting Information). Previous
photophysical characterization of Acd by Szymanska et al. has
shown that Acd emission undergoes a blue shift in nonpolar
environments. Since polycyclic aromatic molecules have been
shown to bind in the helical cleft of CaM, we believe that the
41
(
TIM), and calmodulin (CaM) in E. coli. PAGE gel analysis
and mass spectra confirming incorporation of Acd in these
proteins are given in Supporting Information (Figure S12 and
D
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Journal of the American Chemical Society
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change in Acd emission occurs because pOCNC binding forces
the Acd residue to move from a hydrophobic pocket into
1:1 complex is about half of the emission resulting from direct
excitation of CaM-L113δ at 385 nm). Similar results were
obtained with binding Mcm-pOCNC to CaM-F δ and CaM-
6
1−63
solvent.
While solvatochromic effects at position 41 could
13
be used to monitor peptide binding to CaM, they complicate
the interpretation of FRET data, so we focused our attention
on the other positions.
To demonstrate the use of Acd as a FRET acceptor for Trp
and Mcm, we synthesized variants of pOCNC labeled at the N-
terminus with these fluorophores. The CaM Acd mutants were
each titrated with Trp-pOCNC or Mcm-pOCNC, and
fluorescence emission recorded from 350 to 550 nm while
exciting at 295 nm (Trp) or 325 nm (Mcm). Binding data for
Mcm-pOCNC and CaM-L113δ are shown in Figure 5. We see a
Y100δ (see Figure S19, Supporting Information). As one can see
in Table 1, The excellent quantitative agreement of the
expected and observed EFRET gives us confidence in using the
Mcm/Acd FRET pair to study protein folding.
Table 1. FRET in CaM/Mcm-pOCNC Complexes
a
b
c
CaM mutants
CaM-F13
EQ obsvd
R (Å)
EQ calcd
δ
0.92
0.81
0.98
16
18
13
0.91
0.81
0.97
CaM-Y100
δ
δ
CaM-L113
a
EQ determined from quenching in 1:1 CaM/Mcm-pOCNC complex.
R is an average value calculated from the twenty lowest energy
b
structures in PDB ID 1SYD using the positions of the corresponding
59 c
amino acids.
Φ of 0.098 determined for Mcm-pOCNC in 1:1
D
complex with CaM by measurement of the fluorescence of equimolar
concentrations of free Mcm-pOCNC, and 1:1 CaM/Mcm-pOCNC.
68
Mcm-pOCNC Φ assumed to be 0.18, the value for free Mcm. R0
D
for Mcm/Acd calculated using Φ of 0.098 is 23 Å.
D
Binding of Trp-pOCNC to any of the three CaM mutants
can also be monitored by FRET (see Figure S20/21,
Supporting Information). In this case, no background
subtraction is necessary for superstoichiometric complexes
since Trp has no fluorescence emission above 400 nm.
However, there is substantial direct excitation of Acd upon
irradiation at 295 nm (27% of the emission due to excitation at
Figure 5. Monitoring CaM binding by FRET. Fluorescence spectra of
1
1
μM CaM-L113δ in the presence of 0, 0.1, 0.25, 0.5, 0.75, 1.0, 1.2, or
.5 equiv of Mcm-pOCNC, colored red, orange, yellow, light green,
dark green, aqua, blue, and purple, respectively. The fluorescence
spectrum of 1 μM Mcm-pOCNC alone is shown in dashed gray. All of
these spectra were recorded with excitation at 325 nm. The spectrum
with 1.5 equiv of Mcm-pOCNC is a difference spectrum in which the
emission from 0.5 μM Mcm-pOCNC was subtracted from the
spectrum of the 15 μM Mcm-pOCNC/10 μM CaM-L113δ mixture.
The fluorescence spectrum of 1 μM CaM-L113δ alone with excitation
at 385 nm is shown in black. Inset: Peptide binding monitored by
changes in the emission at 442 nm due to excitation at 325 nm,
normalized to direct Acd excitation at 385 nm. Additional data points
at Mcm-pOCNC/CaM-L113δ ratios of 0.05, 0.15, 0.2, 0.3, 0.35, 0.4,
3
85 nm). Since there is also a possibility of Trp quenching of
Acd emission through PET, we are hesitant to interpret EFRET
data in terms of distances for Trp in the same way that we do
for Mcm. In addition, most proteins contain more than one
Trp, so FRET data would need to be fit to complex models
with more than one donor fluorophore. Regardless, Trp FRET
can still easily be used to quantify protein−protein binding
energies by fitting concentration-dependent data. One of the
protein partners could be WT, provided that it contained at
least one Trp residue. As noted above, Trp and Mcm each have
benefits and drawbacks as Acd FRET donors. Since some
quenching may occur via PET to Trp, we view Mcm as a more
optimal FRET donor. However, many researchers may find
Trp/Acd FRET pairs more useful due to the ease of genetically
encoding doubly labeled proteins.
0
.45, 0.55, 0.6, 0.65, 0.7, 0.8, 0.85, 0.9, 0.95, 1.1, 1.3, and 1.4 are shown.
decrease in Mcm emission and an increase in Acd emission due
to FRET. These data, obtained using 1 μM CaM, can be fit to
obtain a dissociation constant of ≤9 nM. This is slightly higher
than the 3 nM K previously reported for pOCNC, but this
d
may reflect the changes in our system (a shorter peptide than
60
that used in Liu et al., as well as Mcm and Acd labels). The
In addition to singlet energy transfer interactions with
organic chromophores like Trp or Mcm, Acd can undergo
intersystem crossing to donate energy to lanthanide ions such
binding affinity is consistent with low nanomolar K values
d
previously reported for natural and synthetic helical peptides as
well as mutants of CaM that alter the hydrophobic packing of
3+
41
as Eu . While this has previously been demonstrated in short,
synthetic peptides, the ability to genetically encode Acd allows
us to implement the same Eu3+ LRET probes in proteins
64−67
the peptide-binding pocket.
Several features of this experiment are important to note.
First, we can see that binding reaches saturation in the 1:1
complex, as determined by examining the difference spectrum
in which the emission from 0.5 equiv of Mcm-pOCNC was
subtracted from the spectrum of the 1.5:1 Mcm-pOCNC/
CaM-L113δ mixture (purple trace in Figure 5). Second, FRET is
efficient, as one would expect for a complex in which Mcm and
Acd are separated by ∼13 Å. For this separation, the theoretical
3
+
expressed in E. coli. To demonstrate Acd-sensitized Eu
emission, we relied on the precedent for CaM binding a
3+
3+ 69−71
variety of cations, including the lanthanides Tb and Eu .
Since efficient LRET typically requires close contact between
the donor chromophore and lanthanide ion, we chose CaM-
Y100δ as a construct for LRET studies because Y100 is adjacent
to both C-terminal binding sites. Titration of CaM-Y100δ with
3
+
FRET efficiency (E
) is 0.97, which compares well to the
Eu(NO ) revealed a dose-dependent increase in Eu emission
FRET
3 3
measured EFRET of 0.98, determined from the decrease in donor
emission. Third, Mcm and Acd are a robust FRET pair, with
minimal direct excitation of Acd at 325 nm (see red trace in
Figure 5) and strong emission due to FRET (emission in the
at 592 and 615 nm. The 615 nm emission data could be fit to a
two site binding model with K ’s of 28 μM and 154 μM and
d
Hill coefficients of 1.1 and 2.6, respectively. The 615 nm signal
primarily derives from the lower-affinity binding event(s),
E
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Journal of the American Chemical Society
Article
which presumably corresponds to binding at the C-terminus,
other at the dimer interface and they are relatively far (≥13 Å)
from the other Trp and Tyr residues.
particularly to site III, defined by residues D , D , N , Y100,
9
4
96
98
S102, and E105. Previous experiments have found higher affinity
K = 1 μM) binding of lanthanides to this site; the lower
affinity observed here may be due to disruption by the Acd
The single mutants TIM-F δ and TIM-Y101δ were purified,
74
(
and fluorescence spectra were acquired in varying concen-
trations of urea. In the absence of urea, both mutants are
quenched relative to the fluorescence of an equimolar
concentration Acd (urea had no effect on the fluorescence of
d
residue, as studies have found that minor mutations at site III
2
+
72−74
can dramatically decrease Ca affinity.
For higher
free Acd). TIM-F δ is quenched to a greater degree, as one
specificity LRET labeling, one may use lanthanide-binding
74
would expect, since it is in contact with Tyr101 of the other TIM
monomer unit in the folded, dimeric native state of TIM. The
TIM-Y101δ mutant, on the other hand, places no Trp or Tyr in
direct contact with Acd. The two mutants recover comparable
levels of fluorescence upon titration with urea, since quenching
by Tyr101 is relieved as TIM unfolds and dimer dissociation
occurs. In addition to monitoring unfolding by changes in
steady-state fluorescence, we also find it useful to measure
quenching by changes in the fluorescence lifetime of Acd.
Lifetime-based measurements of quenching made by TCSPC
are very valuable, because they are not sensitive to
sequences of the type described by Imperiali and co-
workers.
1
3,75
53
concentration matching like steady-state measurements.
Consistent with our steady state data, we observe a decrease
in Acd lifetime in both TIM-F δ and TIM-Y101δ relative to free
74
Acd: τAvg(free) = 15.8 ns, τAvg(TIM-F δ) = 6.8 ns, and
74
Figure 6. Monitoring Eu3+ Binding to CaM By LRET. Luminescence
emission spectra of 10 μM CaM-Y100δ in the presence of 0, 1, 2, 5, 6, 8,
τAvg(TIM-Y101δ) = 12.7 ns. Lifetime measurements in 0, 2, 3,
and 4 M urea demonstrate that the more dramatic Tyr-specific
quenching in TIM-F δ is relieved by 3 M urea, and that global
1
0, 12, 16, 20, 24, 32, 40, 48, 56 equiv of EuNO , colored dashed red,
3
74
dashed green, dashed blue, red, orange, yellow, chartreuse, light green,
dark green, aqua, light blue, dark blue, light purple, dark purple, and
black, respectively. Spectra recorded with excitation at 385 nm. Inset:
unfolding gives comparable lifetimes for both mutants in 4 M
urea. These experiments show how changes in Acd lifetime, due
either to general quenching phenomena and/or specific
placement near a quenching residue, may be used to monitor
unfolding.
3
+
Eu binding monitored by changes in the emission at 615 nm. Data fit
to two cooperative binding transitions as described in Supporting
Information.
CONCLUSIONS
In many proteins, specific quenching interactions with a
nearby Trp or Tyr may occur, as implicated by our Stern−
Volmer experiments. These could be engineered into either
peptide or protein binding partners, and Acd quenching could
be used to monitor bimolecular interactions. Indeed, we have
shown that this is the case for Acd mutants of TIM. TIM forms
■
The fluorescent unnatural amino acid Acd had previously been
shown to be a useful probe of protein conformational change,
but its use had been limited to synthetic peptides and in vitro
translation. Here, we report an efficient and affordable synthesis
of Acd, and the identification of an Acd-specific variant of the
M. janaschii TyrRS. These developments allow one to easily
produce milligram quantities of Acd-labeled proteins for
biophysical studies. We have characterized the photophysical
properties of Acd, including quenching interactions with select
natural amino acids and FRET interactions with common
fluorophores such as coumarin. We have also demonstrated the
incorporation of Acd into several proteins and used changes in
Acd fluorescence to monitor peptide binding and ion binding as
well as protein folding.
7
6,77
a dimer as shown in Figure 7.
We selected Phe and Tyr
74 101
as positions for Acd incorporation since they approach each
Of course, the use of Acd as a probe of protein structure and
function is not limited to the examples here. Acd quenching or
FRET can also be used to monitor intramolecular conforma-
tional changes, either using a Trp/Acd FRET pair or an Mcm/
Acd pair by labeling a Cys residue with a methoxycoumarin
derivative. LRET applications using the optimized lanthanide-
binding tags developed by the Imperiali group could enhance
the utility of Acd-based LRET.
One of the advantages of our synthesis relative to previously
published methods is its versatility. Some aspects of Acd
photochemistry are nonideal, such as its low extinction
coefficient and blue wavelength emission. In some experiments,
its sensitivity to solvent may also be problematic. We would like
to synthesize analogues of Acd that might exhibit improved
photophysical properties. Fortunately, our synthetic route is
compatible with analogues of Tyr and anthranilate that contain
Figure 7. Triose phosphate isomerase (TIM) unfolding. Left: An
image showing TIM dimer (protomers colored cyan and green) with
Phe74 (orange) and Tyr101 (pink) highlighted in both protomers.
Other Trp and Tyr residues in both protomers are shown in stick
representation and are labeled in the green protomer. Image adapted
7
7
̈
from PDB 1TRE using PyMol (Schrodinger, LLC; New York, NY).
Right: Monitoring TIM unfolding monitored by changes in average
fluorescence lifetime for solutions of 2 μM TIM-F δ (orange), TIM-
74
Y101δ (magenta), or free Acd (blue) in 15 mM HEPES 140 mM KCl
buffer with varying concentrations of urea. Acd lifetime derived from
biexponential fits to the primary data. See Supporting Information for
details.
F
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Journal of the American Chemical Society
Article
electron-withdrawing or electron-donating groups. The result-
(np), 155.3 (np), 148.0 (np), 139.8 (np), 134.2 (+), 131.8 (+), 131.3
(np), 130.4 (+), 122.6 (+), 117.3 (+), 114.2 (+), 112.1 (np), 80.0
ing Tyr analogues should have a “push-pull” chromophore that
78−81
(np), 54.6 (+), 52.4 (+), 51.9 (+), 38.0 (−), 28.5 (+); HRMS (ESI)
is more red-shifted and brighter than the parent Acd.
+
m/z calcd for C H N O [M + H] 429.2000, found 429.2026.
23
28
2
6
Finally, since the AcdRS identified here can also charge
tRNACUA with Brb, we have undertaken crystallography studies
to determine the structural basis for substrate binding. Our
preliminary analysis indicates that Glu65 may be important and
that selectivity toward Acd vs Tyr is conferred by a few
residues. As noted within, this is not surprising for a synthetase
that is permissive of several large amino acids. We are exploring
the permissivity of the AcdRS toward other tricyclic scaffolds
such as xanthones and acridines. We expect that many such
fluorescent amino acids may be incorporable.
(
S)-2-Amino-3-(9-oxo-9,10-dihydroacridin-2-yl) Propanoic
Acid (Acd, 1). Twelve milliliters of 13.5 M sulfuric acid was added
to a flask containing 6 (1.02 g, 2.38 mmol). The flask was then fitted
with a reflux condenser and heated to 115 °C for 16 h in an oil bath.
80 mL of water was then added slowly to the flask and allowed to stir
for 15 min. The reaction was then removed from the hot oil bath and
allowed to cool with stirring. Upon reaching ambient temperature, the
solution was then cooled to 4 °C and allowed to stand for 2 h. During
the 2 h cooling period, 100 g of Dowex ion-exchange resin was made
into a slurry with 1.8 M aqueous sulfuric acid and applied to a flash
chromatography column. The resin was washed with 350 mL of 1.8 M
aqueous sulfuric acid, 2 L of water, 1 L of 1.5 M aqueous ammonium
hydroxide, and 4 L of water. Following these washes, the resin was
dried by passing air through the column. The cooled Acd solution was
EXPERIMENTAL PROCEDURES
■
General Information. L-Tyrosine, thionyl chloride, di-tert-butyl
dicarbonate (Boc anhydride), methyl 2-aminobenzoate, and phenyl-
sepharose CL-4B resin were purchased from Sigma-Aldrich (St. Louis,
MO, USA). N-Phenyl-bis(trifluoromethane sulfonimide) was pur-
chased from Oakwood Chemical (West Columbia, SC, USA).
Eu(NO ) came from Strem Chemicals (Newburyport, MA, USA).
̈
then vacuum filtered on a Buchner funnel to remove precipitated
material and the clarified solution was applied to the washed and dried
ion-exchange resin. The resulting resin slurry was shaken in the
chromatography column for 5 min before the solution was drained.
This solution is then reapplied to the dried resin and shaken for an
additional 5 min. The twice-passed solution was then set aside. The
loaded resin was washed by 4 L of water before the compound of
interest was eluted with 1.45 L of 1.5 M ammonium hydroxide. The
solution was concentrated to 50 mL by rotary evaporation and then
lyophilized to dryness, yielding a crop of Acd as a yellow powder
3
3
E. coli BL21(DE3) cells were purchased from Stratagene (La Jolla, CA,
USA). E. coli ElectroMAX DH10B cells were purchased from
Invitrogen (Grand Island, NY, USA). Milli-Q filtered (18 MΩ)
water was used for all solutions (Millipore; Billerica, MA, USA). All
peptide synthesis reaction vessels (RVs) were treated with Sigmacote
prior to use.
(0.6375 g 2.26 mmol 94.9%). The ion-exchange resin was recycled by
washing with 4 L of water and dried until further use. To maximize
yield, the twice-passed solution was reapplied to washed and dried ion-
exchange resin, and the process was repeated to yield a second crop of
Acd (0.0232 g of 0.082 mmol 3.4%). Total yield for the two columns
was 98.3% and purity was >98% by analytical HPLC. Characterization
DNA sequencing was performed at the University of Pennsylvania
DNA sequencing facility. Matrix-assisted laser desorption ionization
(
MALDI) mass spectra were collected with a Bruker Ultraflex III
MALDI-TOF-TOF mass spectrometer (Billerica, MA). UV/vis
absorbance spectra were obtained with a Hewlett-Packard 8452A
diode array spectrophotometer (currently Agilent Technologies; Santa
3
7
of this compound matched previous reports. Saponification and
polyphosphoric acid (PPA) cyclization procedures are given in
Supporting Information.
1
13
Clara, CA). NMR spectra, H and C, were collected with a Bruker
1
DRX 500 MHz instrument. CDCl was calibrated at δ 7.27 for H and
3
δ 77.2 for 13C. DEPT-135 Carbon NMR notation as follows: no signal
Candidate Synthetase Screening. For screening of Acd
incorporation, protein expressions were performed in DH10B cells
transformed with pBad-sfGFP-150TAG and a synthetase-containing
is designated by (np), a positive signal is designated by (+), and a
negative signal is designated by (−). High-resolution mass spectra
39
pDule vector to produce UAA-sfGFP as previously described.
(
(
HRMS) were obtained on a Waters LC-TOF mass spectrometer
model LCT-XE Premier) using electrospray ionization in positive
Arabinose autoinduction media and method were used. UAAs were
dissolved in sterile water with 1 mol equiv of aqueous NaOH and then
added to the appropriate media to a final concentration of 1 mM.
Negative control cultures of the mutant proteins, containing no UAA,
were grown simultaneously. Cultures at 37 °C were inoculated with
mode.
Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-
((trifluoromethyl)sulfonyl)oxy)phenyl)propanoate (4). Com-
pound 4 was synthesized essentially as described. Characterization
(
3
7,39
1
:100 dilution of saturated noninducing cultures. Standard expressions
matched previous reports.
were performed in duplicate with 0.45 mL of media in a 96-well 2 mL
culture plate with shaking and incubation at 37 °C. Fluorescence
measurements of the cultures were collected 40 h after inoculation
using a BioTek Synergy 2 microplate reader. The emission from 528
nm (20 nm bandwidth) with excitation at 485 nm (20 nm bandwidth).
Samples were prepared by diluting suspended cells directly from
culture 10-fold with phosphate buffer saline. To quantify the protein
yield, DH10B E. coli cells cotransformed with the pBad-sfGFP-
150TAG vector and the machinery plasmid pDule-RS were used to
inoculate 5 mL of noninducing media containing 100 mg/mL
ampicillin (Amp) and 25 mg/mL tetracycline (Tet). The noninducing
media culture was grown to saturation with shaking at 37 °C and 1.5
mL was used to inoculate 150 mL of autoinduction media with 100
mg/mL Amp, 25 mg/mL Tet, and 1 mM Uaa. After 40 h of shaking
with incubation at 37 °C, cells from 100 mL of culture were collected
by centrifugation. The protein was purified using BD-TALON cobalt
ion-exchange chromatography. Purified proteins were checked for
concentration by Bradford protein assay.
(
S)-Methyl 2-((4-(2-((tert-butoxycarbonyl)amino)-3-me-
thoxy-3-oxopropyl)phenyl)amino)benzoate (6). 75 mL of
toluene was degassed with argon for 15 min in a 250 mL flame-
dried round-bottom flask. Tyr derivative 4 (3.000 g, 7.02 mmol) was
added to the flask followed by methyl 2-aminobenzoate (1200 μL, 9.27
mmol). This solution was degassed with argon for 5 min. Then
palladium(II) acetate (0.082 g, 0.365 mmol), and racemic 2,2′-
bis(diphenyl-phosphino)-1,1′-binaphthyl (0.054 g, 0.087 mmol) were
added to the flask. Cesium carbonate (6.88 g, 21.1 mmol) was ground
and added to the flask. The flask was then fitted with a reflux
condenser and heated to 135 °C for 23 h. After the solution was
allowed to cool to ambient temperature, the contents were filtered
through a short plug of silica gel using CH Cl to transfer the material
2
2
to the silica (250 mL) and then ethyl acetate (500 mL) was used to
elute the product. The clarified solution was then concentrated under
reduced pressure. Silica gel flash column chromatography (10% ethyl
acetate in hexanes) afforded 2.759 g (6.44 mmol, 91.7%) of a pale
yellow oil. R = 0.2 in 15% ethyl acetate in hexanes; H NMR (500
1
f
MHz, CDCl ) δ 9.44 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.31 (t, J = 7.9
Hz, 1H), 7.23 (d, J = 8.5 Hz 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.10 (d, J =
Calmodulin Binding Assays. All peptide binding experiments
were conducted in 15 mM HEPES buffer, 140 mM KCl, and 6 mM
3
8
.1 Hz, 1 H), 6.73 (t, J = 7.5 Hz, 1 H), 5.05 (d, J = 7.9 Hz, 1 H), 4.61
CaCl , pH 6.70. Dry peptides and lyophilized protein were brought up
2
−
2
4.57 (m, 1 H), 3.90 (s, 3H), 3.74 (s, 3H), 3.07 (dd, J = 26.5, 5.7 Hz,
in a minimal amount of buffer to make fresh concentrated stock
solutions for each experiment. Protein concentrations were
H), 1.43 (s, 9H); 13C NMR (125 MHz, CDCl ): δ 172.5 (np), 169.0
3
G
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Journal of the American Chemical Society
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determined by (1) use of Thermo Scientific’s Pierce BCA protein
assay kit using known concentrations of bovine serum albumin as the
standard and (2) UV/vis absorbance measurements using Acd
ES00210, NIEHS, NIH to R.A.M.). Instruments were
supported by the NSF and NIH include HRMS (NIH RR-
0
23444) and MALDI-MS (NSF MRI-0820996). We thank Dr.
−1
−1 39
absorbance (ε386 = 5.700 M ·cm ). Mcm-pOCNC concentrations
Simon Berritt of the UPenn/Merck High Throughput
Experimentation (HTE) Laboratory (NSF GOALI CHE-
were determined using 7-methoxycoumarin absorbance (ε = 12 000
3
25
−1
−1 68
M ·cm ). Fluorescence spectra were obtained using a Photon
Technologies International (PTI; Piscataway, NJ, USA) Quantamaster
0
848460) for assistance with reaction screening. The HTE
Laboratory is thankful for the donations of Accelrys Library
Studio and Virscidian Analytical Studio software. We also thank
Dr. Christopher Lanci of the UPenn Biological Chemistry
Resource Center for assistance with automated peptide
synthesis and circular dichroism spectroscopy (supported by
UPenn Laboratory for Research on the Structure of Matter with
NSF MRSEC DMR 0520020, 1120901). L.C.S. thanks
Professor Eric J. Schelter (UPenn) for Eu(NO ) . J.M.G.
4
0 fluorescence spectrometer. For titrations using CaM-L113δ and
Mcm-pOCNC, samples consisted of 1 μM CaM-L113δ and increasing
Mcm-pOCNC concentrations from 0.05 μM to 1.5 μM in 15 mM
HEPES, 140 mM KCl and 6 mM CaCl , pH 6.70. The excitation
wavelength was 325 nm, and emission spectra were collected from 350
to 550 nm. For titrations using CaM-Y100δ and Eu , samples consisted
of 10 μM CaM-Y100δ and increasing Eu(NO ) concentrations from
2
3+
3
3
1
.0 μM to 560 μM in 15 mM HEPES and 140 mM KCl at pH 6.70.
3
3
The excitation wavelength was 386 nm, and emission spectra were
collected from 400 to 650 nm. Instrument settings for these
experiments were 1 nm slit widths at both the excitation and emission
monochromators, 1 nm step size, and 1 s averaging time.
thanks Tom Troxler of the Ultrafast Optical Processes
Laboratory (NIH 9P41GM104605) for assistance with
TCSPC measurements.
TIM Urea Assays. All unfolding experiments were conducted in 15
mM HEPES buffer and 140 mM KCl, pH 6.70 with varying
concentrations of urea. After Ni-NTA column purification, the pure
protein fractions were dialyzed three times against this buffer without
urea. Protein concentrations were determined with a bicinchoninic
acid (BCA) assay kit (Thermo Scientific Pierce) using solutions of
bovine serum albumin (BSA) as standards. For each mutant, solutions
were prepared that contained approximately 10 μM protein, and the
appropriate concentration of urea was established by addition of
various amounts of a 10 M urea, 15 mM HEPES, and 140 mM KCl
buffer pH 6.70. Each sample was prepared in triplicate. Time-resolved
fluorescence measurements were performed using the time-correlated
single photon counting (TCSPC) method. The TCSPC system
consisted of a blue diode laser (PicoQuant GmbH; Berlin, Germany)
generating 10 MHz output pulses at 405 nm, a subtractive double
monochromator with an MCP-PMT (Hamamatsu Photonics R2809U;
Bridgewater, NJ), and a TCSPC computer board (Becker and Hickl
SPC-630; Berlin, Germany). Emission at 450 nm was monitored. All
samples were thermostatted at 25 °C. Data analysis was performed
with FluoFit software (PicoQuant) as described in Supporting
Information.
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ASSOCIATED CONTENT
Supporting Information
■
*
S
Screening of conditions for Pd cross-coupling, additional
synthetic procedures, steady state and lifetime Stern−Volmer
fluorescence quenching experiments, protein mutagenesis,
expression, and characterization, primary fluorescence data for
CaM, TIM, and αS experiments. This material is available free
of charge via the Internet at http://pubs.acs.org. Primary data
used to generate figures and tables have been digitally archived
and can be obtained by emailing the corresponding author.
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AUTHOR INFORMATION
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(18) Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.;
Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohane, R. F.;
Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris,
G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V.
ACS Chem. Biol. 2008, 3, 373−382.
Corresponding Authors
ejpetersson@sas.upenn.edu
ryan.mehl@oregonstate.edu
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