Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase

Article abstract of DOI:10.1021/ja207719f

Tyrosyl radicals (Y?s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (F_nYs, n = 1-4) are promising tools for studying the mechanism of Y? formation and reactivity, as their pK _a values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F₃Y) and demonstrate their ability to charge an orthogonal tRNA with a series of F_nYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate F_nYs site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y?s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with F_nY(s), and the resulting proteins are isolated in good yields. F_nYs incorporated at position 122 of β2, the site of a stable Y? in wild-type RNR, generate long-lived F_nY?s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y₁₂₂(2,3,5)F₃Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y? can accumulate. The distinct EPR properties of the 2,3,5-F₃Y? facilitate spectral subtractions that make detection and identification of new Y?s straightforward.

Full text of DOI:10.1021/ja207719f

COMMUNICATION
pubs.acs.org/JACS
Incorporation of Fluorotyrosines into Ribonucleotide Reductase
Using an Evolved, Polyspecific Aminoacyl-tRNA Synthetase
||
||
Ellen C. Minnihan,†, Douglas D. Young,§, Peter G. Schultz,*,§ and JoAnne Stubbe*,†,‡
†Department of Chemistry and ‡Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,
Massachusetts 02139, United States
§Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S Supporting Information
b
Scheme 1. Working Mechanism for the Long-Range PCET
Pathway in E. coli Class Ia RNRa
ABSTRACT: Tyrosyl radicals (Y s) are prevalent in biolo-
gical catalysis and are formed under physiological conditions
3
by the coupled loss of both a proton and an electron.
Fluorotyrosines (FnYs, n = 1ꢀ4) are promising tools for
studying the mechanism of Y formation and reactivity,
3
as their pKa values and peak potentials span four units and
300 mV, respectively, between pH 6 and 10. In this manu-
script, we present the directed evolution of aminoacyl-tRNA
synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F3Y)
and demonstrate their ability to charge an orthogonal tRNA
with a series of FnYs while maintaining high specificity over Y.
a The current hypothesis holds that protons (blue arrows) move
An evolved aaRS is then used to incorporate FnYs site-
orthogonally to the electron (red arrow) in β2 and co-linearly with
specifically into the two subunits (α2 and β2) of Escherichia
the electron in α2. The mechanism across the α/β interface is unknown.
coli class Ia ribonucleotide reductase (RNR), an enzyme that
There is no direct evidence that W48 and its putative H+ acceptor, D237
participate in long-range PCET during turnover.
,
employs stable and transient Y s to mediate long-range, re-
3
versible radical hopping during catalysis. Each of four con-
served Ys in RNR is replaced with FnY(s), and the resulting
proteins are isolated in good yields. FnYs incorporated at
Ribonucleotide reductase (RNR) catalyzes the formation of all
four 20-deoxynucleotides from the corresponding nucleoside
diphosphates (NDPs) by a mechanism involving protein and
position 122 of β2, the site of a stable Y in wild-type RNR,
3
generate long-lived FnY s that are characterized by electron
3
nucleotide radicals.1 In the class Ia RNRs, a stable diferric-Y
paramagnetic resonance (EPR) spectroscopy. Furthermore,
we demonstrate that the radical pathway in the mutant
Y122(2,3,5)F3Y-β2 is energetically and/or conformationally
modulated in such a way that the enzyme retains its activity
3
cofactor in the β2 subunit initiates catalysis by transiently oxidiz-
ing a cysteine at the active site in the α2 subunit. The mechanism
of radical initiation has been best studied in Escherichia coli RNR
and is believed to involve an unprecedented mechanism of re-
versible long-range (>35 Å) PCET along a pathway of absolutely
but a new on-pathway Y can accumulate. The distinct EPR
3
properties of the 2,3,5-F3Y facilitate spectral subtractions that
3
conserved redox-active amino acids: Y122 T [W48?] T Y356 in
make detection and identification of new Y s straightforward.
3
3
β2 to Y731 T Y730 T C439 in α2 (Scheme 1).5,6 This hypothesis
has been tested by the site-specific incorporation of more than a
half-dozen unnatural amino acids (UAAs) at positions along the
pathway using both expressed protein ligation (EPL)7 and in vivo
nonsense suppression8,9 techniques. Studies in which a series of
fluorotyrosines (FnYs) were incorporated at position 356 of β2
by EPL were particularly informative, as the pH rate profiles of
the resulting proteins indicated that this residue is redox-active,
that proton and electron movement is orthogonal at this position
(Scheme 1), and that an ordered H-bonding network involving
the Y356 phenol is not necessary for catalysis.10,11
xamples of amino acid radicals participating in charge-
E
transfer reactions are prevalent in nature.1 Of these, tyrosyl
radicals (Y s) have been shown to mediate a number of key
3
metabolic transformations, including O2 evolution, fatty acid oxida-
tion, peroxide disproportionation, and prostaglandin synthesis.2
The thermodynamics of Y oxidation require that proton transfer
(PT) accompanies electron transfer (ET) at physiological pH, a
process that may occur by either a stepwise or coupled (PCET)
mechanism. To investigate the mechanism of Y formation and
3
Y -mediated catalysis in metalloenzymes, a method by which
3
FnYs incorporated in place of other conserved Ys along the
pathway would be comparably informative. However, the EPL
technology used to generate Y356FnY-β2s cannot be readily
extended to positions in the protein interior. While other strategies
the native Y may be subtly perturbed is needed. A series of
3
N-acetylfluorotyrosinamides (N-Ac-FnY-NH2s) were character-
ized in solution and found to have phenolic pKa values ranging from
5.6 to 8.4 and peak potentials ranging from 705 to 968 mV.3,4 This
series of Y analogues provides a means of systematically modulat-
ing the chemical properties governing both the PT and ET
events.
Received: August 15, 2011
Published: September 13, 2011
r
2011 American Chemical Society
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COMMUNICATION
for incorporation of FnYs exist, namely, global incorporation4,12
or site-specific insertion of photocaged FnYs,13 these techniques are
applicable to mono- and di-FnYs only and are minimally useful in
RNR, as the former lacks specificity and the latter leads to com-
plications arising from photolysis.14
Herein, a robust method for in vivo site-specific incorporation
of a series of FnYs is reported. This method requires evolution of
an orthogonal aminoacyl-tRNA synthetase (aaRS)ꢀtRNA pair
and has been successfully used to incorporate over 70 UAAs into
E. coli, Saccharomyces cerevisiae, and mammalian proteins in
response to a nonsense (or “blank”) codon.15,16 Originally, we
sought to evolve an aaRS specific for 2,3,5-trifluorotyrosine
(2,3,5-F3Y, 1), as previous data suggested that its redox potential
is sufficiently increased relative to Y at appropriate pHs that its
incorporation at position 356 of β2 changes the rate-determining
step in RNR from a conformational change17 to PCET.11 Three
fluorine substituents together with the low phenolic pKa (6.4)
were expected to allow the evolution of an RS selective for the
phenolate of 1 over the Y phenol.
To incorporate 1 into proteins selectively, an orthogonal
Methanococcus jannaschii aaRSꢀtRNA pair that suppresses the
amber stop codon (TAG) was evolved to encode this amino acid
uniquely by a double-sieve selection process based on cellular
viability.16 A library of TyrRS mutants was generated by rando-
mizing eight positions near the active site based on the known
structure of Tyr-bound MjTyrRS,18 including those residues
closest to the aromatic ring (Y32, L65, and H70) and those
involved in H-bonding to the Y phenol (Y32 and D158). The
library was subjected to three positive and negative selection
cycles, after which individual candidates were isolated and
assessed for viability in the presence and absence of 1 at different
chloramphenicol (Cm) concentrations. The selection resulted in
two primary clones, E3 (Y32L, L65G, H70N, F108F, Q109Q,
D158S, I159Y, L162H) and E11 (Y32H, L65Y, H70G, F108Y,
Q109A, D158N, I159I, L162R), that conferred Cm resistance
(>150 μg/mL) only in the presence of 1 (Figure S1 and Table S1
in the Supporting Information). Given the mutations and the
structure,18 we tried to understand the basis for recognition of
1 by the aaRSs (Figure S2). We had predicted that the negative
charge on the phenolate of 1 would be the basis for discrimina-
tion against Y and anticipated the introduction of positively
charged side chains in the mutants. E3 contains a L162H
mutation, but the protonation state of this residue is unknown.
E11 contains a single basic side chain (L162R); however, a crystal
structure of the p-acetylphenylalanine RS containing the same
mutation revealed that the R side chain was directed away from
the active site.19 Y32 and/or D158 are mutated in both 2,3,5-
F3Y-RSs to residues that could conceivably behave as H-bond
donors to the phenolate of 1. The substitution of Gly at
either position 65 (E3) or 70 (E11) may enlarge the substrate
binding pocket. Thus, there exists minimal evidence indicating
that the observed discrimination occurs on the basis of the
phenolate. Instead, it appears that the fluorine substitutents
provide the key recognition elements. The introduction and
retention of many polar side chains provide opportunities for the
three F atoms to participate in multipolar interactions of the
nature CꢀF HꢀN/O or CꢀF CdO.20 In view of the
Figure 1. Polyspecificity of FnY-RSs. (A) Y analogues that screened
positive for GFP incorporation. (B) GFP fluorescence assay of the two
evolved aaRSs (blue, E3; red, E11) based on suppression of GFPY151TAG
in the presence of 1 mM UAA.
is of general interest, given the large number of fluorinated
pharmaceuticals.20
The aaRS hits were cloned into the previously described
pEVOL system21 for enhanced expression of proteins containing 1.
We confirmed the selective incorporation of 1 into proteins by aaRSs
E3 and E11 using pET-GFPY151TAG, a plasmid that encodes
a C-terminally His6-tagged green fluorescent protein (GFP).15 Full-
length fluorescent protein is observed only when the TAG codon is
suppressed by an amino-acylated tRNA. E3 afforded 10 mg/L of
purified GFP, while E11 yielded 4 mg/L. Both aaRSs incorporated 1
into GFP with an observed mass of 27 759 Da (27 758 Da expected;
see Figure S3). In the absence of 1, low levels of phenylalanine
incorporation could be detected for E3; however, this background
incorporation was not detected in the presence of 1 (a property
observed with several previously evolved aaRSs).22a No back-
ground incorporation was detected for E11 in the absence of 1.
Recent experiments have shown that some aaRSs exhibit a
degree of polyspecificity.22 This feature arises from the lack of
other UAAs in the selection medium to eliminate synthetases
that can recognize them. To determine whether these 2,3,5-F3Y
aaRSs are polyspecific, they were screened against a diverse
library of UAAs using a GFP fluorescence assay.22a Of the ∼80
UAAs in the library, both aaRSs were capable of aminoacylating
3-fluorotyrosine (2) and 3-chlorotyrosine (8) (Figure S4). The
results of this first screen indicated that the aaRSs exhibit some
polyspecificity for other halogenated Ys, prompting a second
GFP screen on Ys with a narrower range of substitutions
(including 3ꢀ7, 9, and 10). The evolved aaRSs were capable
of incorporating all members of the FnY series [3-FY (2), 2,3-F2Y
(3), 3,5-F2Y (4), 2,3,6-F3Y (5), and 2,3,5,6-F4Y (6)] as well
as several other halogenated analogues (Figure 1). The incorporation
of each UAA was confirmed by LC/MS analysis of the mutant
GFPs (Figure S5). Interestingly, several of the analogues (2ꢀ5)
were incorporated into GFP at higher levels than 1, thus the
evolved aaRSs are henceforth called “FnY-RSs.” LC/MS analysis
of E. coli lysate indicated that reduced cellular uptake is one
reason for the low incorporation of 6 relative to the other FnYs.
3 3 3
3 3 3
sensitivity of these interactions to distance and geometry, it
is impossible to speculate on the specific atoms involved without
a high-resolution structure. Crystallization attempts to provide a
better understanding of the basis of Fꢀprotein interactions in
the 2,3,5-F3Y-RSs are underway. The nature of these interactions
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COMMUNICATION
The FnY-RS E3 was then used to incorporate FnYs at positions
730 and 731 of the RNR α2 subunit (Scheme 1). These residues
were recently shown to be sites of transient Y formation during
3
radical propagation by substitution of 3-aminotyrosine (NH2Y)8
and 3-nitrotyrosine (NO2Y)9 at these positions using similar
technology. Cells were transformed with pEVOL-FnY-RS-E3 and
pET-nrdAY730TAG, encoding for α with a N-His6 tag and a stop
codon at the position of Y730,
23 and grown in the presence of 1.
SDS-PAGE of whole cells after induction revealed a ∼1:1 ratio
of full-length and truncated proteins (Figure S6). The desired
protein, Y730(2,3,5)F3Y-α2, was isolated by Ni-NTA chroma-
tography to give 10 mg of protein/g of cell paste (∼50 mg/L of
culture). An analogous protocol was used to incorporate 3, 4,
and 5 at position 730 of α2. These Y730FnY-α2s were purified in
yields of 2.5ꢀ3 mg/g (Table S2). Consistent with the GFP data
(Figure 1), the yield of protein containing 6 was 10-fold lower
than for the other FnYs. The 730-optimized protocol was then
used to incorporate 1 and 4 at position 731 of α2 with
comparable protein yields. Finally, the expression protocol was
used with minor modification to incorporate 1 at position 356 of
β2, and the protein was purified (2 mg/g).
Figure 2. X-band EPR spectra of E. coli wt Y122 (black), 3,5-F2Y122 (dark-
3
3
blue), and 2,3,5-F3Y (cyan). Spectra were recorded at 77 K and normalized for
3
radical concentration. Simulations (red) were performed using EasySpin.26
X-band EPR analysis of the purified proteins at 77 K indicated
that the as-isolated 3,5-F2Y and 2,3,5-F3Y mutants contained
0.2 and 0.02 FnY122 /β2, suggesting that the FnY122 s are
3
3
Incorporation of FnYs at position 122 of β2, the site of the
inherently less stable than native Y122 . Higher radical content
3
stable Y (t1/2 ≈ 4 days, 4 °C) in the wild type (wt), was of
3
was obtained for 2,3,5-F3Y122 mutants (0.4ꢀ0.9 2,3,5-F3Y122 /β2)
3
mechanistic interest. The reduction of Y122 occurs concomitant
by in vitro reconstitution of the cofactor.27 Both the 3,5-F2Y122
3
3
with oxidation of C439 in α2 (Scheme 1), yet the intermediates of
and the 2,3,5-F3Y122 were stable for more than 30 min at 4 °C.
The EPR spectra of the putative 3,5-F2Y and 2,3,5-F3Y at position
122 of β2 (Figure 2, blue and cyan) are quite distinct from those
3
radical propagation in the wt enzyme are kinetically masked by
rate-limiting conformational changes.17 Thus, changes to Y122
3
3
3
or any of the pathway residues are undetectable by rapid bio-
physical techniques. Furthermore, even in those mutant RNRs
in which radicals can be trapped and/or conformational gat-
ing can be partially lifted, observation and identification of new
radicals by X-band EPR spectroscopy is complicated by their
extensive spectral overlap. Recently, site-specific insertion of
NO2Y at position 122 of β2 was described. Reaction of apo-
of FnY s generated on the corresponding free amino acids by UV
3
photolysis.4 The spectra of the latter are relatively featureless, with
line broadening arising from a large distribution of dihedral angles
for the β-methylene protons. In contrast, structural constraints
imposed by the protein give rise to FnY122 spectra with well-
3
defined couplings to the fluorine nuclei (150ꢀ180 MHz) and one
of the β-methylene protons (36ꢀ48 MHz). Spectral simulations
(Figure 2, red; Table S3) were conducted using the parameters for
Y
122NO2Y-β2 with Fe2+ and O2 led to the assembly of a diferric-
NO2Y122 cluster.24 Use of the potent oxidant NO2Y122 as a
3-FY122 as an initial reference.4 High-field EPR/ENDOR analysis
3
3
3
radical initiator decoupled the ET from the rate-limiting confor-
of the FnY s is ongoing (Myers, Minnihan, Stubbe, and Britt).
3
mational change and allowed the first detection of a transient Y
Nucleotide reductase assays of Y122(2,3,5)F3Y-β2 revealed the
mutant has 30% of the wt β2 activity (scaled for radical). This
result suggests that introduction of 2,3,5-F3Y at position 122
slightly perturbs the radical propagation energetics and/or the
conformational changes that gate it, but not to the extent that it
prevents multiple catalytic turnovers. The reactivity of this
3
on the pathway.24,25 However, the high redox potential of NO2Y
relative to Y (Δ ≈ 200 mV, pH 7) introduced complications into
the system, namely, the short half-life of NO2Y122 (t1/2 ≈ 40 s,
3
25 °C) and the mutant’s inability to perform multiple turnovers.
Given these results, we sought to modulate the properties of
position 122 more subtly using FnYs. In view of the known
perturbation of the phenolic pKa at this position (>2.5 units),9
UAAs 1 and 4 should be protonated at physiological pH and are
predicted to be <10 mV harder and ∼50 mV easier to oxidize
mutant was further investigated by EPR spectroscopy. Y122
-
(2,3,5)F3Y-β2 was mixed on ice with wt-α2, CDP (substrate),
and ATP (allosteric effector) under single-turnover conditions in
an EPR tube and quenched in a dry ice/acetone bath at 20 s. The
spectrum of the reaction mixture (Figure 3, black) is a composite
than Y , respectively. Using pEVOL-FnY-RS-E3 and pBAD-
3
of the spectra of two species: 2,3,5-F3Y122 (pink) and a new
nrdBY122TAG, encoding untagged β2 with a stop codon at
3
position 122,24 the proteins Y122(2,3,5)F3Y-β2 and Y122
-
radical (blue). Subtraction of the former from the composite
was done using the fluorine hyperfine couplings, which occur in
unobstructed regions of the spectrum. Calculation of the double
integral intensities of the two species indicated that the new radical
constitutes 25% of the total spin. The new radical is assigned
(3,5)F2Y-β2 were expressed and isolated by anion-exchange
chromatography in yields of g30 mg/g. The two mutants were
characterized by UVꢀvis and EPR spectroscopy to determine
whether they contained any FnY . The as-isolated wt subunit
3
as Y356 on the basis of additional EPR experiments in which
contains 1.2 Y /β2, with a sharp diagnostic absorbance at 411 nm
3
3
the pathway was blocked at specific positions by insertion of a redox-
inert F. Indeed, when Y122(2,3,5)F3Y-β2wasreactedwithY731F-α2,
a very similar new radical was formed in identical yield (Figure S7).
A reaction with the double mutant Y122(2,3,5)F3Y/Y356F-β2 and
wt-α2 gave no evidence of a new radical. The assignment was
further substantiated by the strong similarity of this radical to the
and a doublet X-band EPR spectrum dominated by coupling to
one of the β-methylene protons (Figure 2, black). As-isolated
Y122(3,5)F2Y-β2 and Y122(2,3,5)F3Y-α2 hadabsorptionsatλmax
=
396 and 404 nm, respectively, consistent with the values deter-
mined for 3,5-F2Y and 2,3,5-F3Y generated by photooxidation
3
3
of benzophenone-FnY-OMe dipeptides.4
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COMMUNICATION
Department of Energy (P.G.S.). D.D.Y. acknowledges an NIH Ruth L.
Kirchstein Postdoctoral Fellowship (F32CA144213). We thank the
Gao Lab (Boston College) for providing Fmoc-F4Y.
’ REFERENCES
(1) Stubbe, J.; van der Donk, W. A. Chem. Rev. 1998, 98, 705.
(2) (a) Barry, B. A.; el-Deeb, M. K.; Sandusky, P. O.; Babcock, G. T. J.
Biol. Chem. 1990, 265, 20139. (b) Gupta, A.; Mukherjee, A.; Matsui, K.;
Roth, J. P. J. Am. Chem. Soc. 2008, 130, 11274. (c) Zhao, X.; Suarez, J.; Khajo,
A.; Yu, S.; Metlitsky, L.; Magliozzo, R. S. J. Am. Chem. Soc. 2010, 132, 8268.
(d) Tsai, A. L.; Kulmacz, R. J. Arch. Biochem. Biophys. 2010, 493, 103.
(3) Kim, K.; Cole, P. A. J. Am. Chem. Soc. 1998, 120, 6851.
(4) Seyedsayamdost, M. R.; Reece, S. Y.; Nocera, D. G.; Stubbe, J.
J. Am. Chem. Soc. 2006, 128, 1569.
Figure 3. EPR spectrum of the reaction of Y122(2,3,5)F3Y-β2, wt-α2, CDP,
and ATP quenched at 20 s. Subtraction of the 2,3,5-F3Y122 contribution
3
(pink) from the reaction spectrum (black) gives a new radical (blue). Inset:
(5) Uhlin, U.; Eklund, H. Nature 1994, 370, 533.
(6) Stubbe, J.; Nocera, D. G.; Yee, C. S.; Chang, M. C. Y. Chem. Rev.
2003, 103, 2167.
overlay of the new radical with Y356 formed by Y122NO2Y-β2 (gray).
3
one observed when NO2Y122 was used as a radical initiator
(7) (a) Yee, C. S.; Seyedsayamdost, M. R.; Chang, M. C. Y.; Nocera,
D. G.; Stubbe, J. Biochemistry 2003, 42, 14541. (b) Chang, M. C. Y.; Yee,
C. S.; Nocera, D. G.; Stubbe, J. J. Am. Chem. Soc. 2004, 126, 16702.
(c) Seyedsayamdost, M. R.; Stubbe, J. J. Am. Chem. Soc. 2006, 128, 2522.
(8) Seyedsayamdost, M. R.; Xie, J.; Chan, C. T.; Schultz, P. G.;
Stubbe, J. J. Am. Chem. Soc. 2007, 129, 15060.
(9) Yokoyama, K.; Uhlin, U.; Stubbe, J. J. Am. Chem. Soc. 2010, 132, 8385.
(10) Yee, C. S.; Chang, M. C. Y.; Ge, J.; Nocera, D. G.; Stubbe, J.
J. Am. Chem. Soc. 2003, 125, 10506.
3
(Figure 3 inset).24 Extensive evidence suggests that the new
25
radical in the NO2Y mutant is primarily located at Y356
.
While it is necessary to determine the kinetic competence of the
new radical, observation of a putative Y356 is promising in two regards.
3
First, the ability to observe an on-pathway Y (while maintaining
3
significant catalytic activity) will have important mechanistic implica-
tions. Investigations to determine what properties of F3Y allow
observation of this new radical are underway. We anticipate deriving
additional insight into the long-range PCET mechanism by modulat-
ing the driving force and phenolic pKa at positions on the pathway in a
stepwise fashion. Similarly, FnYs may be utilized for mechanistic studies
of ET and/or PT in model proteins and native enzymes.
(11) Seyedsayamdost, M. R.; Yee, C. S.; Reece, S. Y.; Nocera, D. G.;
Stubbe, J. J. Am. Chem. Soc. 2006, 128, 1562.
(12) Rappaport, F.; Boussac, A.; Force, D. A.; Peloquin, J.; Brynda, M.;
Sugiura, M.; Un, S.; Britt, R. D.; Diner, B. A. J. Am. Chem. Soc. 2009, 131, 4425.
(13) Wilkins, B. J.; Marionni, S.; Young, D. D.; Liu, J.; Wang, Y.; Di
Salvo, M. L.; Deiters, A.; Cropp, T. A. Biochemistry 2010, 49, 1557.
(14) Incorporation of o-nitrobenzyl-FnYs at position 730 of α2 was
previously attempted. The location of this residue in the protein interior
resulted in inefficient decaging. Additionally, recovery of protein activity
after photolysis was poor, likely as a result of side reactions with
neighboring redox-active amino acids.
Additionally, this result demonstrates the utility of FnY s as
3
spectroscopic handles. The width of the FnY X-band EPR spectra
3
and the presence of distinct fluorine hyperfine couplings, which give
rise to features in the low- and high-field spectral regions, allow
accurate subtraction of FnY s from other g ≈ 2 radicals (e.g., Y ,
3
3
[β-2H2]Y , NH2Y , NO2Y ).8,24 Thus, FnY s afford the first
(15) Young, T. S.; Schultz, P. G. J. Biol. Chem. 2010, 285, 11039.
(16) (a) Xie, J.; Schultz, P. G. Methods 2005, 36, 227. (b) Liu, C. C.;
Schultz, P. G. Annu. Rev. Biochem. 2010, 79, 413.
3
3
3
3
opportunity to identify low concentrations of radical species that
would otherwise go undetected in complicated reaction mixtures.25
(17) Ge, J.; Yu, G.; Ator, M. A.; Stubbe, J. Biochemistry 2003, 42, 10071.
(18) Kobayashi, T.; Nureki, O.; Ishitani, R.; Yaremchuk, A.; Tukalo, M.;
Cusack, S.; Sakamoto, K.; Yokoyama, S. Nat. Struct. Biol. 2003, 10, 425.
(19) Turner, J. M.; Graziano, J.; Spraggon, G.; Schultz, P. G. J. Am.
Chem. Soc. 2005, 127, 14976.
(20) (a) Muller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881. (b)
Zhou, P.; Zou, J.; Tian, F.; Shang, Z. J. Chem. Inf. Model. 2009, 49, 2344.
(21) Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G. J. Mol. Biol.
2010, 395, 361.
(22) (a) Young, D. D.; Young, T. S.; Jahnz, M.; Ahmad, I.; Spraggon,
G.; Schultz, P. G. Biochemistry 2011, 50, 1894. (b) Miyake-Stoner, S. J.;
Refakis, C. A.; Hammill, J. T.; Lusic, H.; Hazen, J. L.; Deiters, A.; Mehl,
R. A. Biochemistry 2010, 49, 1667.
(23) Minnihan, E. C.; Seyedsayamdost, M. R.; Uhlin, U.; Stubbe, J.
J. Am. Chem. Soc. 2011, 133, 9430.
FnY s may prove similarly useful in high-field EPR and ENDOR
3
studies. For proteins that do not use radicals, FnYs may be
incorporated as probes of structure and/or local environment for
19F NMR studies.28 Thus, the application of FnYs as spectroscopic
probes may be extended to a broad biophysical research community.
’ ASSOCIATED CONTENT
S
Supporting Information. Methods; FnY-RS selection;
b
structure of MjTyrRS active site; GFP polyspecificity screen;
LC/MS of FnY-GFPs; expression/purification of FnY-RNRs;
EPR reaction spectra for Y122(2,3,5)F3Y-β2 with blocked mu-
tants. This material is available free of charge via the Internet at
http://pubs.acs.org.
(24) Yokoyama, K.; Uhlin, U.; Stubbe, J. J. Am. Chem. Soc. 2010,
132, 15368.
(25) Yokoyama, K.; Smith, A. A.; Corzilius, B.; Griffin, R. G.; Stubbe,
’ AUTHOR INFORMATION
Corresponding Author
schultz@scripps.edu; stubbe@mit.edu
J. Submitted.
(26) Stoll, S.; Schweiger, A. J. Magn. Reson. 2006, 178, 42.
(27) Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.;
Edmondson, D. E.; Stubbe, J. Methods Enzymol. 1995, 258, 278.
(28) Li, C.; Wang, G. F.; Wang, Y.; Creager-Allen, R.; Lutz, E. A.;
Scronce, H.; Slade, K. M.; Ruf, R. A.; Mehl, R. A.; Pielak, G. J. J. Am.
Chem. Soc. 2010, 132, 321.
Author Contributions
These authors contributed equally.
’ ACKNOWLEDGMENT
This work was supported by NIH Grant GM29595 (J.S.) and
Grant DE-FG03-00ER46051 from the Division of Materials Sciences,
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dx.doi.org/10.1021/ja207719f |J. Am. Chem. Soc. 2011, 133, 15942–15945