Biosynthesis of a fluorescent protein with extreme Pseudo-stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore

Article abstract of DOI:10.1021/ja1099787

A novel kind of fluorescent protein relying on the intramolecular interplay between two different fluorophores, one of chemical origin and one of biological origin, was developed. The fluorescent non-natural amino acid l-(7-hydroxycoumarin-4-yl)ethylglyci

Full text of DOI:10.1021/ja1099787

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Biosynthesis of a Fluorescent Protein with Extreme Pseudo-Stokes
Shift by Introducing a Genetically Encoded Non-Natural Amino Acid
outside the Fluorophore
Sebastian M. Kuhn, Marina Rubini, Michael A. M€uller, and Arne Skerra*
Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl f€ur Biologische Chemie, Technische Universit€at M€unchen, Emil-
Erlenmeyer-Forum 5, 85350 Freising-Weihenstephan, Germany
S Supporting Information
b
at the same position of the uvGFP variant via amber suppression
ABSTRACT: A novel kind of fluorescent protein relying on
the intramolecular interplay between two different fluoro-
phores, one of chemical origin and one of biological origin,
was developed. The fluorescent non-natural amino acid
L-(7-hydroxycoumarin-4-yl)ethylglycine was site-specifi-
cally incorporated into the recombinant enhanced cyan
fluorescent protein (eCFP) at a permissible surface position
∼20 Å away from the protein fluorophore using amber
suppression in Escherichia coli with an engineered cognate
Methanococcus jannaschii tRNA synthetase. The resulting
eCFPCou exhibited almost quantitative intramolecular
F€orster resonance energy transfer (FRET) between its
two fluorophores, showing brilliant cyan emission at 476
nm upon excitation in the near-UV at 365 nm (a wavelength
easily accessible via conventional laboratory UV sources), in
contrast to its natural counterpart. Thus, this fluorescent
protein with unprecedented spectroscopic properties re-
veals an extreme apparent Stokes shift of ∼110 nm between
the absorption wavelength of the coumaryl group and the
emission wavelength of eCFP.
employing an orthogonal aminoacyl-tRNA synthetase (aaRS)/
tRNA pair, leading to another biosynthetic FP with unique
spectral properties.4-6
With regard to the design of novel FPs, the latter approach in
principle also allows insertion of spectroscopically active side
chains at positions outside the central fluorophore, thus indir-
ectly modulating its excitation and/or emission behavior. In this
report, we describe the incorporation of a coumaryl residue at a
permissible surface position of eCFP that forms a highly efficient
F€orster pair with the inner fluorophore, yielding an FP with an
extreme apparent Stokes shift of ∼110 nm between the absorp-
tion wavelength of the coumaryl group and the emission
wavelength of eCFP.
The fluorescent amino acid L-(7-hydroxycoumarin-4-
yl)ethylglycine (1) (Figure 1) was previously incorporated into
proteins via amber suppression with an engineered aaRS.7 In its
phenolate state, 1 exhibits strong fluorescence at ∼450 nm with a
high quantum yield (QY) of 0.63 upon excitation at 360 nm.
Here we show that the spectral properties of 1 also make it a
suitable intramolecular donor for eCFP by means of F€orster
resonance energy transfer (FRET).8,9 FRET is a quantum-
mechanical phenomenon that occurs when two fluorophores in
sufficient spatial proximity (<100 Å) show spectral overlap
between the emission band of the shorter-wavelength fluoro-
phore (the “donor”) and the absorption band of the longer-
wavelength fluorophore (the “acceptor”). As the so-called
F€orster effect is strongly distance-dependent, it is often applied
in biochemistry as a kind of macromolecular ruler.8,10
While initially applied in conjunction with chemical labeling
of biomolecules, FRET between different GFPs—as biological
gene products—has also been exploited, thus opening its use in
cell biology.11-13 However, a problem of FPs for many
practical applications is their typically inefficient excitation in
the near-UV spectral range. The most commonly used eGFP
and, in particular, eCFP show poor brightness when illumi-
nated with a typical laboratory UV light source or a laser at 366
nm. To address this general limitation, we sought to evoke an
intramolecular FRET effect by combining the fluorescence
activity of a natural FP with the spectrally matching fluores-
cence of a non-natural amino acid incorporated during its
biosynthesis. As an initial attempt, we chose eCFP as the FRET
acceptor for 1 because its absorption maximum in the range of
Since the first application of the recombinant green fluores-
cent protein (GFP) from Aequorea victoria,1 the class of visibly
fluorescent proteins (FPs) has become an invaluable molecular
tool in cell and developmental biology, biophysics, and biotech-
nology. Numerous engineered versions of GFP itself as well as
orthologues from other (mainly marine) organisms have been
described.2 From alteration of the substitution pattern or in-
traprotein environment of the 4-(p-hydroxybenzylidene)imida-
zolin-5-one core fluorophore, which autocatalytically forms from
three amino acids as part of the polypeptide chain, a whole series
of FPs with a broad spectrum of absorption and emission
characteristics can result. Several attempts have been made to
substitute the aromatic side chain at position 66 of GFP
(originally Tyr), whose π-electron system is conjugated to the
heterocyclic core group (see Figure 1) and strongly influences its
spectroscopic properties.
However, the chemical space of the 20 natural amino acid
building blocks is limited, and early on there was a desire to
introduce other types of side chains. For example, 4-aminotryp-
tophan has been incorporated at this position of the enhanced
cyan fluorescent protein (eCFP) via metabolic pressure to yield a
“golden FP”.3 Furthermore, O-methyl-L-tyrosine was introduced
Received: November 6, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ja1099787 J. Am. Chem. Soc. 2011, 133, 3708–3711
|

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Figure 1. Molecular structures of the fluorophores used in this study.
(left) Three-dimensional model of eCFPCou, which carries the fluor-
escent amino acid 1 at the surface-exposed position 39, based on the
crystal structure of eCFP (PDB entry 2WSN). (right) Chemical
structures of (bottom) the eCFP fluorophore 4-[(1H-indol-3-
yl)methylidene]imidazolin-5-one (5; λex = 434 nm, λem = 476 nm)
and (top) the fluorescent non-natural amino acid L-(7-hydroxycoumar-
in-4-yl)ethylglycine (1; λex = 360 nm, λem = 450 nm), which together
can form a F€orster pair. In the structure of 5, R1 and R2 denote the N-
and C-terminal polypeptide segments, respectively.
430-450 nm almost perfectly coincides with the 7-hydroxy-
coumarin emission maximum (Figure 2). Although eCFP has
a moderate QY of 0.4 for 476 nm emission,14 its high excit-
ation coefficient of 32 500 M-1 cm-1 at 434 nm is ideal for
efficient (radiationless) absorption of donor fluorescence
energy (i.e., FRET).
1 was chemically synthesized according to a published
procedure7,15 with an improved purification by crystalliza-
tion (see the Supporting Information). To achieve cotranslational
incorporation of 1 into eCFP, its coding region was expressed in
Escherichia coli using a recently developed one-plasmid expression
Figure 2. Spectroscopic analysis of the novel FP. (A) Normalized
absorption and fluorescence spectra of 1 [Abs(1), Flu(1)] and eCFP
[Abs(eCFP), Flu(eCFP)]. The major absorption band of eCFP shows
considerable overlap with the fluorescence spectrum of 1, thus fulfilling a
prerequisite for efficient FRET. (B) Fluorescence spectra of 1, eCFP,
and eCFPCou (2.2 μM each) upon excitation at 365 and 434 nm. Upon
excitation at 365 nm, the coumaryl donor emission in eCFPCou is almost
completely quenched (comparison of blue and orange lines, respec-
tively, at 450 nm) whereas its acceptor-group emission is dramatically
increased (orange line at 476 nm) in comparison with that of the
unmodified eCFP (green). Notably, upon excitation at 434 nm, both
eCFP and eCFPCou show identical high emission (dashed green and
orange lines, respectively).
system6 thatalso carried the genes foranaaRSspecific to17 andfor
Tyr
CUA
a cognate amber suppressor tRNA . For site-directed incor-
poration of the foreign amino acid, an amber stop codon (TAG)
was introducedatthepositionofTyr39 inthe eCFPreading frame,
and the Strep-tag II16 was appended at the C-terminus to allow
affinity purification. In the crystal structure of eCFP17 (PDB entry
2WSN), the side chain of Tyr39 is located in a loop on the surface
of the β-can motif at a distance of ∼20 Å from the central eCFP
fluorophore, which is in sufficient proximity to ensure efficient
FRET (Figure 1).
From a 1 L culture of E. coli BL21 harboring this system in rich
medium supplemented with 1, ∼0.1 mg of the recombinant
protein (dubbed eCFPCou) was obtained after purification via
Strep-Tactin affinity chromatography and polishing by anion-
exchange and size-exclusion chromatography. High protein
purity was confirmed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and electrospray ionization
mass spectrometry (ESI-MS) (Figure 3). In particular, the mass
spectrum revealed a unique prominent peak at 27959.8 Da,
which closely matches the calculated value of 27959.4 Da. This
demonstrated essentially quantitative incorporation of 1 instead
of the original Tyr residue, apart from full maturation of the
central eCFP fluorophore, which is accompanied by a loss of
H2O and H2, as well as complete processing of the N-terminal
Met residue.
The biosynthetic eCFPCou was investigated by collecting its
absorption and fluorescence spectra at pH 9.0 (Figure 2 and
Figure S4 in the Supporting Information) and comparing them
with those of the synthetic fluorescence donor compound 1 and
the natural bacterially produced fluorescence acceptor eCFP.
The isolated donor and acceptor fluorescence spectra showed
the expected maximal absorption and fluorescence wavelengths,
respectively, around 360 and 450 nm for 1 and 434 and 476 nm
for eCFP. Notably, eCFPCou exhibited an absorption spectrum
corresponding closely to the theoretical absorption of an equi-
molar mixture of 1 and eCFP (Figure 2 and Figure S4).
However, upon excitation at 365 nm, the anticipated donor
fluorescence in eCFPCou was efficiently quenched (Figure 2B,
orange line at 450 nm) whereas a bright acceptor fluorescence
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Figure 3. Analysis of the bacterially produced and purified fluorescent
proteins eCFP and eCFPCou by ESI-MS and Coomassie-stained SDS-
PAGE (inset). For unlabeled eCFP (calcd mass 27877.3 Da), only a
Figure 4. Eppendorf tubes filled with 1 μM solutions of 1, eCFP or
eCFPCou, all excited with a conventional UV hand lamp at 366 nm. The
strongly enhanced fluorescence of eCFPCou vs eCFP is visible, and the
fluorescence is clearly red-shifted in comparison with that of 1.
minute signal (amounting to less than 2.6% of the intensity for eCFPCou
)
was detectable in the mass spectrum, thus confirming essentially quanti-
tative incorporation of 1 instead of the natural Tyr residue at position 39.
in the significantly shorter range of 200 ps to 1.6 ns,19 thus
preventing an unfavorable orientation factor. Furthermore, a
calculation of the critical F€orster transfer distance R0 (using an
orientation factor κ2 = 2/3)20 based on the excitation and emission
spectra collected for the eCFP fluorophore and 1, respectively,
yielded a value of 45 Å, which is considerably larger than their
spatial distance in the biosynthetic hybrid protein (Figure 1) and
hence explains the high efficiency of this process.
As a result of the almost complete intramolecular energy
transfer in the folded protein, our eCFPCou can be regarded as a
novel type of FP with a large pseudo-Stokes shift of ∼110 nm,
which contrasts with the much lower shift of ∼40 nm for the
natural eCFP (Figure 2A). Until now, such a pseudo-Stokes shift
above 100 nm could be achieved almost only with tandem dyes
or quantum dots.21
Our new FP should be useful not only for biophysical and
spectroscopic studies. As one advantage, the fluorescence of
eCFPCou upon irradiation with a standard 366 nm UV lamp
available in most laboratories is easily detectable by the naked
eye, whereas the natural eCFP shows only very faint fluorescence
under such conditions (Figure 4). More generally, our approach
should be applicable to other combinations of fluorescent
proteins with non-natural fluorescent amino acids, providing
novel reagents with spectroscopic properties tailored for various
purposes.
at 476 nm appeared (Figure 2B, orange line). In fact, the
coumaryl fluorescence was suppressed by a factor of 17, as
estimated from a comparison of the stationary peak fluores-
cence at 450 nm with that of an equimolar mixture of 1 with
eCFP, while the concomitant eCFPCou emission had a peak
intensity more than 6-fold higher than the one of eCFP upon
excitation at 365 nm (Figure 2B, comparison of orange and
green lines). Still, it was also possible to excite eCFPCou at 434
nm, again giving rise to the typical eCFP fluorescence at 476 nm
with unchanged QY.
The observed strong quenching of the hydroxycoumaryl
donor group in eCFPCou indicated potent intramolecular
FRET. The FRET efficiency in biochemical systems is usually
quantified according to the change in the ratio of acceptor to
donor stationary peak fluorescence upon excitation at the
donor’s excitation wavelength (here 365 nm). In the case of
eCFPCou, this ratio showed a remarkable 9.3-fold increase, from
a value of 0.70 measured for a mixture of the two isolated
fluorophores to 6.5 in the biosynthetic protein (using donor
and acceptor emission wavelengths of 452 and 476 nm,
respectively). Since eCFP exhibits a double-peak fluorescence
spectrum,14 the ratio of acceptor to donor emission even
increased to 16.3 when the acceptor emission at 505 nm was
probed, because the spectral overlap with 1 in the equimolar
mixture serving as the reference decreased at this higher
fluorescence wavelength. On the other hand, the fluorescence
of eCFP was only ∼30% lower at this longer wavelength than at
its first emission maximum at 476 nm.
’ ASSOCIATED CONTENT
S
The highly efficient F€orster transfer in our biosynthetic hybrid
protein is remarkable and deserves further discussion. The
7-hydroxycoumarylethyl amino acid employed here as the donor
has three intra-side-chain rotational degrees of freedom (CR-Cβ,
Cβ-Cγ, Cγ-Cδ). With its solvent- exposed position at the
protein surface, it is unlikely to be trapped in an orientation
unsuitable for FRET with the central eCFP fluorophore upon
consideration of the roles of fluorescence anisotropy and polariza-
tion. The fluorescence lifetime of 7-hydroxy-4-methylcoumarin is
∼15 ns for the anionic form.18 On the other hand, the rotational
relaxation times of amino acid side chains in unfolded proteins lie
Supporting Information. Details of plasmid design, re-
b
combinant protein production and purification, synthesis and
characterization of the non-natural amino acid, absorption and
fluorescence measurements, MS analyses, and pH titration data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
skerra@tum.de
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’ NOTE ADDED AFTER ASAP PUBLICATION
In the version of this Communication published ASAP February
22, 2011, Cβ-Cγ was omitted from the list of three intra-side-chain
rotational degrees of freedom for the 7-hydroxycoumarylethyl
amino acid. The corrected version was published February 25,
2011.
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dx.doi.org/10.1021/ja1099787 |J. Am. Chem. Soc. 2011, 133, 3708–3711