Detection of dihydrofolate reductase conformational change by fret using two fluorescent amino acids

Article abstract of DOI:10.1021/ja403007r

Two fluorescent amino acids, including the novel fluorescent species 4-biphenyl-l-phenylalanine (1), have been incorporated at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes [1 and l-(7-hydroxycoumarin-4-yl)ethylglycine (2)] were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for Foerster resonance energy transfer (FRET) measurements on the basis of their estimated separation (17-18 A) and the expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (1 at position 17; 2 at position 115) and DHFR II (2 at position 17; 1 at position 115) were both catalytically competent. However, DHFR II containing the potentially rotatable biphenylphenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of 1, energy transfer to 2, and emission by 2 at 450 nm. However, the energy transfer was substantially more efficient in DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration-dependent diminution of the emission observed at 450 nm for DHFR II but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function and in a fashion that enables sensitive monitoring of changes in DHFR conformation.

Full text of DOI:10.1021/ja403007r

Communication
pubs.acs.org/JACS
Detection of Dihydrofolate Reductase Conformational Change by
FRET Using Two Fluorescent Amino Acids
Shengxi Chen,† Nour Eddine Fahmi,† Lin Wang,‡ Chandrabali Bhattacharya,† Stephen J. Benkovic,*,‡
and Sidney M. Hecht*,†
†Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe,
Arizona 85287, United States
‡Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
S
* Supporting Information
although the use of dye−quencher pairs has enabled the
measured distance to be ∼23 Å.3 The fluorophores are typically
ABSTRACT: Two fluorescent amino acids, including the
novel fluorescent species 4-biphenyl-L-phenylalanine (1),
have been incorporated at positions 17 and 115 of
dihydrofolate reductase (DHFR) to enable a study of
conformational changes associated with inhibitor binding.
Unlike most studies involving fluorescently labeled
proteins, the fluorophores were incorporated into the
amino acid side chains, and both probes [1 and L-(7-
hydroxycoumarin-4-yl)ethylglycine (2)] were smaller than
fluorophores typically used for such studies. The DHFR
large polycyclic aromatic molecules, and they are generally
attached to the macromolecule by flexible tethers, giving them
conformational freedom independent of conformational changes
in the macromolecule.
We recently described the use of dihydrofolate reductase
(DHFR) containing two pyrenylalanines to measure protein
dynamics via the observation of excimer formation.4 The
distance changes measured in DHFR were on the order of
several angstroms.4,5 In an effort to develop a complementary
technique enabling the measurement of conformational changes
in proteins over somewhat longer distances, we explored the use
of FRET. The smaller distances of interest in comparison with
those typically measured by FRET argued for the attachment of
the dyes close to the protein backbone with fewer degrees of
conformational freedom. This in turn would necessitate the use
of acceptors and donors sterically similar to the side chains of
proteinogenic amino acids. The use of fluorescent acceptors
provides qualitative assurance of energy transfer through the
longer-wavelength emission from the acceptor fluorophore6 and
has been reported to have higher sensitivity than dye−quencher
systems and to enable ratiometric analysis, which is generally
superior to intensity-based measurements.7,8
Proteins containing fluorescent amino acids have been
reported,9 and a few studies have employed proteins with two
such amino acids to record large changes in the proximity of the
fluorescent amino acids resulting from protein backbone
cleavage10 or extensive reorganization of protein structure.11 In
the present work, the fluorescent amino acids 4-biphenyl-L-
phenylalanine (1) and L-(7-hydroxycoumarin-4-yl)ethylglycine
(2)12 were used to explore more subtle conformational changes
in Escherichia coli DHFR. These amino acids were of interest
because of the relatively small sizes and complementary shapes of
their side chains. 1 is a para-substituted phenylalanine having two
biphenyl linkages, which should permit some conformational
mobility for inclusion within folded protein structures. 2 has a
bicyclic side chain whose dimensions do not differ dramatically
from those of tryptophan, and it has been reported to have a high
fluorescence quantum yield and to be sensitive to pH and solvent
polarity.13 Critically, the excitation and emission maxima for 1
positions were chosen as potentially useful for Forster
̈
resonance energy transfer (FRET) measurements on the
basis of their estimated separation (17−18 Å) and the
expected change in distance along the reaction coordinate.
Also of interest was the steric accessibility of the two sites:
Glu17 is on the surface of DHFR, while Ile115 is within a
folded region of the protein. Modified DHFR I (1 at
position 17; 2 at position 115) and DHFR II (2 at position
17; 1 at position 115) were both catalytically competent.
However, DHFR II containing the potentially rotatable
biphenylphenylalanine moiety at sterically encumbered
position 115 was significantly more active than DHFR I.
Irradiation of the modified DHFRs at 280 nm effected
excitation of 1, energy transfer to 2, and emission by 2 at
450 nm. However, the energy transfer was substantially
more efficient in DHFR II. The effect of inhibitor binding
was also measured. Trimethoprim mediated concentra-
tion-dependent diminution of the emission observed at
450 nm for DHFR II but not for DHFR I. These findings
demonstrate that amino acids containing small fluoro-
phores can be introduced into DHFR with minimal
disruption of function and in a fashion that enables
sensitive monitoring of changes in DHFR conformation.
orster resonance energy transfer (FRET) has been utilized
̈
Fextensively to monitor conformational changes in macro-
molecules such as nucleic acids and proteins1 as well as
intermolecular association between macromolecules by measur-
ing changes in the efficiency of energy transfer between a donor
and acceptor:2 the fluorophore donor is excited by irradiation,
and the absorbed energy is transferred to the (fluorescent or
quenching) acceptor in a nonradiative process. Distance
measurements made by FRET typically range from 41 to 73 Å,
Received: March 25, 2013
© XXXX American Chemical Society
A
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Journal of the American Chemical Society
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were found to be at 280 and 345 nm, respectively, while the
comparable values for 2 are 345 and 450 nm. This suggested that
it should be possible to selectively excite the 4-biphenyl-L-
phenylalanine moiety in a modified DHFR containing both
species by irradiation at 280 nm and to observe fluorescence from
the coumarin moiety at 450 nm due to FRET (Scheme 1).
phenylalanyl-tRNACCCG was used to suppress the CGGG codon,
while L-(7-hydroxycoumarin-4-yl)ethylgycinyl-tRNACUA was
employed for UAG codon suppression. In the synthesis of 4-
biphenyl-L-phenylalanyl-tRNACCCG (Scheme 2), the key step
Scheme 2
Scheme 1. Strategy Employed for Incorporation of 1 and 2
into DHFR at Positions 17 and 115, respectively
was Suzuki coupling of 4-biphenylboronic acid and protected p-
iodophenylalanine (4) in the presence of Pd(PPh3)4, which
afforded N-Boc-4-biphenyl-L-phenylalanine methyl ester (5) in
99% yield. Subsequent replacement of the Boc protecting group
with an N-pentenoyl group afforded 6 in 51% yield, and
replacement of the methyl ester with a cyanomethyl ester gave N-
(4-pentenoyl)-4-(1′,1″-biphenyl-4′-yl)-L-phenylalanine cyano-
methyl ester (7) in 60% yield. 7 was then treated with 5′-
phosphoro-2′-deoxycytidylyl[3′→5′]adenosine (pdCpA),17 af-
fording aminoacylated dinucleotide 8. Incubation of this
dinucleotide with an abbreviated tRNACCCG-COH transcript
lacking the 3′-terminal nucleotides C and A in the presence of T4
RNA ligase18 afforded 4-biphenyl-L-phenylalanyl-tRNACCCG; the
course of the ligation reaction was monitored by polyacrylamide
gel electrophoresis under acidic conditions.19 The preparation of
L-(7-hydroxycoumarin-4-yl)ethylglycinyl-tRNACUA employed
the same overall strategy; the synthesis of the requisite
aminoacyl-pdCpA intermediate 11 was carried out as shown in
Scheme S1 in the Supporting Information (SI) by modification
of a reported12 procedure.
The amino acid residues chosen for substitution in DHFR
were Glu17 and Ile115, which were estimated to be 17.7 Å apart
and potentially could undergo a change in relative distance of
∼1.1 Å along the collective reaction coordinate.14 The calculated
FRET R0 value for 1 and 2 was 22 Å. Additionally, as may be
appreciated readily from the crystal structure of DHFR (Figure
1), Glu17 is at the enzyme surface and has been shown to tolerate
The first modified DHFR to be synthesized was DHFR I
containing 1 at position 17 and 2 at position 115 (Figure S1 in
the SI). Also prepared were the two DHFRs containing only one
of the two modified amino acids at the same position. The
modified DHFRs were purified by successive chromatography on
Ni-NTA and then DEAE-Sepharose columns. The ability of
these modified enzymes to effect the reduction of dihydrofolate
to tetrahydrofolate was studied to ensure that the isolated
proteins were properly folded. Substitution of position 17 with 1
was well-tolerated, reducing the activity to 76% relative to the
wild-type enzyme (Table S1 and Figure S2). In contrast, the
introduction of 2 at position 115 reduced the consumption of
NADPH to 23% relative to the wild-type enzyme. The doubly
modified DHFR was 20% as active as the wild-type enzyme.
The singly and doubly modified DHFRs were then employed
to determine whether FRET between the two fluorescent amino
acids could be observed. Upon irradiation at 280 nm, the singly
modified DHFR containing 1 at position 17 exhibited an
emission spectrum centered at 345 nm (Figure 2, green curve).
In contrast, the singly modified DHFR containing 2 at position
115 failed to emit significantly at any wavelength when irradiated
at 280 nm (black curve) but gave a strong emission at 450 nm
Figure 1. Structure of wild-type E. coli DHFR (PBD entry 1RA1), with
Glu17 shown in green and Ile115 in blue. Met16, which is spatially close
to the Glu17 side chain (≤4 Å), is shown in red, and Ala7, Trp22, Leu24,
Thr113, and Ala117, which are close to the Ile115 side chain (≤4 Å), are
shown in magenta.
the attachment of a fluorescence quencher,15 while Ile115 is
within a folded region of the protein where changes in side-chain
structure might be expected to affect the protein folding to
varying degrees. Nonetheless, it was anticipated that structural
changes could be made without dramatically affecting the
structure or function of DHFR.
The strategy employed to prepare the modified DHFRs is
illustrated for DHFR I in Scheme 1. Plasmids expressing the
mRNA for DHFR were modified to contain the four-base codon
CGGG16 in lieu of the Glu17 codon GAA, and the nonsense
codon UAG in place of the Ile115 codon AUC. 4-Biphenyl-L-
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two emission peaks centered at 345 and 450 nm. However, in this
case the emission at 450 nm was the stronger of the two. This
established both the greater FRET efficiency of DHFR II relative
to DHFR I and the value of ratiometric analysis in establishing
the relative FRET efficiencies.8 Essentially the same results were
obtained at pH 7.0, but the fluorescence emission was
dramatically diminished at pH 6.0 (Figure S6).
The FRET efficiency E depends on both the distance between
the fluorophores and the dipole orientation factor (κ2). In the
present case, the FRET donor and acceptor do not rotate freely,
so dipole orientation can be important to E. We suggest that 2
can be accommodated less well at position 115 than 1, which has
rotatable biphenyl linkages, and that the resulting distortion in
the structure of DHFR results in the lower FRET and enzymatic
efficiencies of DHFR I compared with DHFR II.20
The ability of the modified DHFRs to monitor changes in
protein conformation was studied by adding the DHFR inhibitor
trimethoprim (TMP). When 0.5 μM DHFR I was treated with
excess TMP, the emission intensities at 345 and 450 nm
decreased in a concentration-dependent fashion (Figure S9). In
contrast, when 0.5 μM DHFR II was treated with TMP, the
intensity of the emission at 345 nm remained constant while that
at 450 nm decreased in a concentration-dependent fashion
(Figure 4), reflecting a decrease in E as a consequence of TMP
Figure 2. Fluorescence of modified DHFRs (0.5 μM) measured at pH
8.0: modified DHFR containing 1 at position 17 with λex = 280 nm
(green); DHFR I containing 1 at position 17 and 2 at position 115 with
λex = 280 nm (red); modified DHFR containing 2 at position 115 with
λex = 280 nm (black) and λex = 340 nm (cyan).
when irradiated at 340 nm (cyan curve). This demonstrated that
irradiation at 280 nm in the doubly substituted DHFR should
effect excitation only of the 4-biphenyl-L-phenylalanine moiety.
When irradiated at 280 nm, doubly modified DHFR I produced
emission peaks at 345 and 450 nm (red curve), with the longer
wavelength emission being of lesser intensity. This established
the successful FRET from excited 1 to 2.
Also prepared using an analogous strategy was isomeric DHFR
II having 2 at position 17 and 1 at position 115. The singly
modified DHFRs having one or the other of these amino acids
were also prepared. Experiments illustrating activation of the
suppressor tRNAs as well as the synthesis and purification of the
proteins are shown in Figures S3−S5, respectively. Interestingly,
the single modification of DHFR with amino acid 2 at position 17
or amino acid 1 at position 115 had essentially no effect on the
activity of DHFR (Table S1 and Figure S2). Doubly substituted
DHFR II retained 50% of the activity of the wild-type enzyme.
The use of DHFR II in a FRET experiment gave results
qualitatively similar to those obtained with DHFR I (Figure 3).
Irradiation at 280 nm to excite the DHFR containing 1 at
position 115 gave a single emission centered at 345 nm. Similar
excitation of the DHFR containing 2 at position 17 gave minimal
emission at 450 nm. Excitation of doubly modified DHFR II gave
Figure 4. Effect of the DHFR inhibitor trimethoprim on the
fluorescence emission spectrum of 0.5 μM modified DHFR II
containing 2 at position 17 and 1 at position 115 following excitation
at 280 nm. Shown are emission spectra of DHFR II alone (red) and after
addition of (blue) 2, (purple) 4, and (green) 6 μM TMP.
addition.20 Thus, for DHFR II, the change in protein structure
occasioned by the addition of TMP can be monitored
conveniently by FRET. The binding of TMP and numerous
other DHFR inhibitors is known to produce only modest
changes in the structure of DHFR,21,22 far smaller than those
ordinarily studied by FRET. Thus, the strategy employed here
involving the careful placement of multiple small fluorescent
amino acids within a protein holds promise for the monitoring of
small conformational changes, such as those that occur during
the catalytic cycle.
It may be noted that the use of suppressor tRNAs activated
with fluorescent amino acids to introduce fluorophores into
proteins has some important advantages relative to other
techniques for producing fluorescently labeled proteins. For
example, post-translational derivatization of cysteine residues is
not position-selective, affording mixtures of products as well as
unmodified cysteine residues that failed to undergo derivatiza-
Figure 3. Fluorescence of modified DHFRs (0.5 μM) measured at pH
8.0 with λex = 280 nm: singly modified DHFR containing 1 at position
115 (green); DHFR II containing 2 at position 17 and 1 at position 115
(red); singly modified DHFR containing 2 at position 17 (black).
C
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Journal of the American Chemical Society
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fluorescence intensity can also be due to changes in the local
environment of the donor or adventitious quenching induced by
species commonly encountered in experimental settings, such as oxygen,
iodine and acrylamide. See: Phillips, S. R.; Wilson, L. J.; Borkman, R. F.
Curr. Eye Res. 1986, 5, 611.
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tion with the fluorophore. The latter is also a potential problem
with proteins prepared using unnatural amino acids having
unique functional groups that can undergo chemoselective
modification with fluorophores following translation.23 In
comparison, the use of amino acids with fluorescent side chains
small enough to permit efficient utilization by the ribosome
enables uniform, site-specific labeling to be verified.24
In conclusion, we have demonstrated the specific incorpo-
ration of two fluorescent amino acids into E. coli dihydrofolate
reductase at sites anticipated to undergo a limited change in
distance from each other along the reaction coordinate. One of
the sites (Glu17) was known from earlier work to tolerate the
introduction of a fluorescence quencher without significant loss
of enzyme function, while the other (Ile115) was known from
crystallographic studies to be more constrained sterically. DHFR
I containing fluorescent amino acids 1 and 2 at positions 17 and
115, respectively, retained catalytic competence but exhibited an
80% reduction in the rate of NADPH oxidation. In contrast, the
introduction of 2 at position 17 had no effect on the rate of
NADPH oxidation, nor did the substitution of 1 at position 115.
Doubly modified DHFR II with 2 at position 17 and 1 at position
115 retained 50% of the activity of wild-type DHFR. This verified
our hypothesis concerning the need to choose the appropriate
amino acid for introduction at position 115 to achieve optimal
fluorescent labeling. While both doubly modified DHFRs
emitted light at 450 nm when the 4-biphenyl-L-phenylalanine
moiety was excited at 280 nm, DHFR II exhibited much more
efficient FRET than DHFR I. Additionally, only DHFR II
exhibited a measurable, dose-dependent change in FRET when
treated with the inhibitor trimethoprim.
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ASSOCIATED CONTENT
* Supporting Information
Methods and additional data. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
sjb1@psu.edu; sid.hecht@asu.edu
■
Notes
The authors declare no competing financial interest.
(20) The involvement of Trp22 in the FRET behavior of DHFR I was
ruled out by introducing Phe at this position. This modified DHFR I had
the same TMP affinity as DHFR I and exhibited essentially the same
FRET behavior upon TMP addition (Figure S7). The absence of direct
effects of TMP on the fluorescence emission was verified by studying
DHFR analogues having 1 or 2 at position 17 (Figure S8). Anisotropy
emission measurements verified the restricted rotational freedom of 1
and 2 at positions 17 and 115 (Table S2).
ACKNOWLEDGMENTS
This study was supported by the NIH (GM 092946).
■
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D
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