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A monomeric red fluorescent protein
Robert E. Campbell*, Oded Tour*†, Amy E. Palmer*, Paul A. Steinbach*†, Geoffrey S. Baird*, David A. Zacharias*†‡,
and Roger Y. Tsien*†§¶
Departments of *Pharmacology and §Chemistry and Biochemistry, and †Howard Hughes Medical Institute, University of California at San Diego,
9500 Gilman Drive, La Jolla, CA 92093
Contributed by Roger Y. Tsien, April 23, 2002
he red fluorescent protein cloned from Discosoma coral
(DsRed or drFP583) (1) holds great promise for biotechnology and cell biology as a spectrally distinct companion or
substitute for the green fluorescent protein (GFP) from the
Aequorea jellyfish (2). GFP and its blue, cyan, and yellow variants
have found widespread use as genetically encoded indicators for
tracking gene expression and protein localization and as donor兾
acceptor pairs for f luorescence resonance energy transfer
(FRET). Extending the spectrum of available colors to red
wavelengths would provide a distinct label for multicolor tracking of fusion proteins, and together with GFP (or a suitable
variant) would provide a FRET donor兾acceptor pair that should
be superior to the currently preferred cyan兾yellow pair (3).
However, the evolution of DsRed from a scientific curiosity to
a generally applicable and robust tool has been hampered by
several critical problems, including a slow and incomplete maturation and obligate tetramerization (4). Most previous attempts
to address the rate and兾or extent of maturation of DsRed (5, 6),
including the commercially available DsRed2 (CLONTECH),
have provided only modest improvements. However, an engineered variant of DsRed, known as T1 (see Fig. 1A), has recently
become available and effectively solved the problem of the slow
maturation (7). Another approach to overcoming these shortcomings has been to continue the search for DsRed homologues
in sea coral and anemone, an approach that has yielded several
red-shifted tetramers (8, 9). The more fundamental problem of
tetramerization, however, has yet to be overcome. The only
published progress toward decreasing the oligomeric state of a
red fluorescent protein involved an engineered DsRed homologue, commercially available as HcRed1 (CLONTECH), which
was converted to a dimer with a single interface mutation (10).
Although HcRed1 has the additional benefit of being 35 nm
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T
www.pnas.org兾cgi兾doi兾10.1073兾pnas.082243699
red-shifted from DsRed, it is limited by a rather low extinction
coefficient (20,000 M⫺1䡠cm⫺1) and quantum yield (0.015) (11).
A variety of techniques have confirmed that DsRed is an
obligate tetramer in vitro (4, 12) and in living cells (4), but for the
researcher who wants to image the subcellular localization of a
red fluorescent chimera the question remains to what extent will
fusing tetrameric DsRed to the protein of interest affect the
location and function of the latter? There have been several
published reports (3, 13), and many unpublished anecdotal
communications, in which DsRed chimeras have been described
as forming intracellular aggregates. The consensus is that a
monomeric form of DsRed will be essential if it is to ever reach
its full potential as a genetically encoded red fluorescent tag (14).
In this article we report the directed evolution and preliminary
characterization of a monomeric red f luorescent protein
(mRFP). Although further refinements of this protein are
desirable and likely, it already provides an independent alternative to GFP in the construction of fluorescently tagged fusion
proteins.
Materials and Methods
Mutagenesis and Screening. DsRed, amplified from vector
pDsRed-N1 (CLONTECH) or T1 (provided by B. S. Glick,
Univ. of Chicago) in pRSETB (Invitrogen) (4), was used as the
template for introduction of the I125R mutant with the QuickChange kit (Stratagene). Error-prone PCR was performed as
described (15). Semirandom mutations at multiple distant locations were introduced by overlap extension PCR (16) with
multiple fragments. Briefly, 2–4 pairs of sense and antisense
oligonucleotides (Invitrogen or GenBase, San Diego), with
semidegenerate codons at positions of interest, were used for
PCR amplification of the DsRed template with Pfu polymerase
(Stratagene) in individual reactions. The resulting overlapping
fragments were gel-purified (Qiagen, Valencia, CA) and recombined by overlap extension PCR with Pfu or Taq polymerase
(Roche Molecular Biochemicals). Full-length genes were digested with BamHI兾EcoRI (New England Biolabs) and ligated
into pRSETB with T4 ligase (New England Biolabs). Chemically
competent Escherichia coli JM109(DE3) were transformed and
grown overnight on LB兾agar at 37°C and maintained at room
temperature for weeks if necessary. LB兾agar plates were
screened by using a cooled charge-coupled device camera as
described (17), and the digital images were processed with
METAMORPH software (Universal Imaging, West Chester, PA).
All screening was done with 470-nm (40-nm bandwidth), 540-nm
(30-nm bandwidth), or 560-nm (40-nm bandwidth) excitation
filters and 530-nm (40-nm bandwidth), 575-nm (long pass), or
610-nm (long pass) emission filters. Colonies of interest were
Abbreviations: DsRed, red fluorescent protein from Discosoma; GFP, green fluorescent
protein; EGFP, enhanced GFP; FRET, fluorescence resonance energy transfer; mRFP, monomeric red fluorescent protein; Cx43, connexin43.
Data deposition: The sequences reported in this paper have been deposited in the GenBank
database (accession nos. AF506025, AF506026, and AF506027).
‡Present address: Merck Research Laboratories, 3535 General Atomics Court, San Diego, CA
92121.
¶To
whom reprint requests should be addressed. E-mail: [email protected].
PNAS 兩 June 11, 2002 兩 vol. 99 兩 no. 12 兩 7877–7882
BIOCHEMISTRY
All coelenterate fluorescent proteins cloned to date display some
form of quaternary structure, including the weak tendency of
Aequorea green fluorescent protein (GFP) to dimerize, the obligate
dimerization of Renilla GFP, and the obligate tetramerization of the
red fluorescent protein from Discosoma (DsRed). Although the
weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically
encoded fusion tag. We present here the stepwise evolution of
DsRed to a dimer and then either to a genetic fusion of two copies
of the protein, i.e., a tandem dimer, or to a true monomer
designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which
initially crippled the resulting protein, but red fluorescence could
be rescued by random and directed mutagenesis totaling 17
substitutions in the dimer and 33 in mRFP1. Fusions of the gap
junction protein connexin43 to mRFP1 formed fully functional
junctions, whereas analogous fusions to the tetramer and dimer
failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living
cells. In addition, the excitation and emission peaks of mRFP1, 584
and 607 nm, are ⬇25 nm red-shifted from DsRed, which should
confer greater tissue penetration and spectral separation from
autofluorescence and other fluorescent proteins.
cultured overnight in 2 ml of LB supplemented with ampicillin.
Bacteria were pelleted by centrifugation and imaged to ensure
that the protein was expressed well in culture. For fast maturing
proteins a fraction of the cell pellet was extracted with B-per II
(Pierce), and complete spectra were obtained. DNA was purified
from the remaining pellet by QIAprep spin column (Qiagen) and
submitted for sequencing. To determine the oligomeric state of
DsRed mutants, a single colony of E. coli was restreaked on
LB兾agar and allowed to mature at room temperature. After 2
days to 2 weeks the bacteria were scraped from the plate,
extracted with B-per II, and analyzed (not boiled) by SDS兾
PAGE (BioRad), and the gel was imaged with a digital camera.
Construction of Tandem Dimers and Constructs for Mammalian Cell
Expression. To construct tandem dimers, dimer2 in pRSETB was
amplified in two separate PCRs. In the first reaction, a 5⬘ BamHI
and a 3⬘ SphI site were introduced, whereas in the second
reaction a 5⬘ SacI and a 3⬘ EcoRI site were introduced. The
construct was assembled in a four-part ligation containing the
digested dimer2 genes, a synthetic linker with phosphorylated
sticky ends, and digested pRSETB. The linkers used included a
nine-residue linker (RMGTGSGQL), a 12-residue linker (GHGTGSTGSGSS), a 13-residue linker (RMGSTSGSTKGQL),
and a 22-residue linker (RMGSTSGSGKPGSGEGSTKGQL).
For expression in mammalian cells, DsRed variants were amplified from pRSETB with a 5⬘ primer that encoded a KpnI
restriction site and a Kozak sequence. The PCR product was
digested, ligated into pcDNA3, and used to transform E. coli
DH5␣. To construct the connexin43 (Cx43) DsRed fusions,
Cx43 was first amplified with a 3⬘ primer encoding a sevenresidue linker ending in a BamHI site. The construct was
assembled in a three-part ligation containing KpnI兾BamHIdigested Cx43, BamHI兾EcoRI-digested enhanced GFP (EGFP),
and digested pcDNA3. For all other fusion proteins (Cx43-T1,
-dimer2, -tdimer2(12), and -mRFP1) the gene for the fluorescent
protein was ligated into the BamHI兾EcoRI digested Cx43-GFP
vector.
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Protein Production and Characterization. DsRed variants were expressed as described (17). All proteins were purified by Ni-NTA
chromatography (Qiagen) and dialyzed into 10 mM Tris, pH 7.5
or PBS supplemented with 1 mM EDTA. All biochemical
characterization experiments, including the determination of
extinction coefficients, analytical ultracentrifugation, and absorption and fluorescence spectra, were performed as described
(4). The maturation time course and pH sensitivity were determined on a Safire 96-well plate reader with monochromators
(TECAN, Männedorf, Switzerland). All photobleaching measurements were performed in microdroplets under paraffin oil
(4) with a Zeiss Axiovert 35 fluorescence microscope equipped
with a ⫻40 objective and a 540-nm (25-nm bandpass) excitation
filter that delivered 4.5 W兾cm2 of light.
Fig. 1. Graphical representation of the tetramer, dimer, and monomer of
DsRed based on the x-ray crystal structure of DsRed (21). Residues 1–5 were not
observed in the crystal structure (Protein Data Bank identification 1G7K) but
have been arbitrarily appended for the sake of representation. The DsRed
chromophore is represented in red, and the four chains of the dimer are
labeled following the convention of Yarbrough et al. (21). (A) The tetramer of
DsRed with all residues mutated in T1 indicated in green for external residues
and blue for those internal to the -barrel. (B) The AC dimer of DsRed with all
mutations present in dimer2 represented as in A and the intersubunit linker
present in tdimer2(12) shown as a dotted line. (C) The monomer of DsRed with
all mutations present in mRFP1 represented as in A. This figure was produced
with MOLSCRIPT (27).
7878 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.082243699
Mammalian Cell Imaging and Microinjection. HeLa cells were transfected with DsRed variants or Cx43-DsRed fusions in pcDNA3
through the use of Fugene 6 (Roche Diagnostics). Transfected
cells were grown for 12 h to 2 days in DMEM at 37°C before
imaging with a Zeiss Axiovert 35 fluorescence microscope with
cells in glucose-supplemented Hanks’ balanced salt solution at
room temperature. Individual cells expressing Cx43 fused to a
DsRed variant, or contacting nontransfected cells for control
experiments, were microinjected with a 2.5% solution of lucifer
yellow (Molecular Probes). Images were acquired and processed
with the METAF LUOR software package.
Results
Evolution of a Dimer of DsRed. Our basic strategy for decreasing the
oligomeric state of DsRed was to replace key dimer interface
Campbell et al.
Table 1. Summary of spectroscopic data
Excitation
maximum, nm
Emission
maximum, nm
Extinction coefficient
per chain, M⫺1䡠cm⫺1
Fluorescence
quantum yield
Fast
Slow
pKa
t0.5 for
maturation
at 37°C
558
555
552
552
584
583
584
579
579
607
57,000
35,000
60,000
120,000
44,000
0.79
0.51
0.69
0.68
0.25
0.23
0.15
0.36
0.31
7.2
0.022
0.012
0.018
0.014
0.16
4.7
4.8
4.9
4.8
4.5
⬃10 h
⬍1 h
⬃2 h
⬃2 h
⬍1 h
residues with arginine. When the targeted residue interacts with
the identical residue of the dimer partner through symmetry, the
high energetic cost of placing two positive charges in close
proximity should disrupt the interaction. Such a strategy disrupted the weak tendency of Aequorea GFP to dimerize, without
any deleterious effects on GFP maturation or brightness (18).
Initial attempts to break apart the DsRed AC interface (see Fig.
1 A) with the single mutations T147R, H162R, and F224R
consistently gave nonfluorescent proteins (19). The AB interface, however, proved somewhat less resilient and could be
broken with the single mutation I125R to give a poorly red
fluorescent dimer that suffered from an increased green component and required more than 10 days to fully mature (19).
To reconstitute the red fluorescence of DsRed–I125R, we
subjected the protein to iterative cycles of evolution. Within each
cycle, the protein was randomly mutagenized to find sequence
locations that affected the maturation and brightness of the
protein, and then expanded libraries of mutations at those
positions were created and recombined to find optimal permutations (see Fig. 6, which is published as supporting information
on the PNAS web site, www.PNAS.org). This strategy resulted in
modest progress toward rescuing DsRed–I125R, but our focus
quickly turned to the fast tetramer T1 (7) when we discovered
that introduction of the I125R mutation into this protein resulted
in a dimer that matured in only a few days, similar to our best
DsRed dimers at that time. Targeting those positions that had
helped rescue DsRed–I125R resulted in dramatic improvements
in our first-generation library. Continuing with our directed
evolution strategy for a total of four generations (see additional
Discussion, Table 2, Table 3, and primer lists, which are published as supporting information on the PNAS web site) we
eventually produced our best dimeric variant, which we have
designated dimer2 (Fig. 1B). Of the 17 mutations in dimer2,
eight are internal to the -barrel (N42Q, V44A, V71A, F118L,
K163Q, S179T, S197T, and T217S), three are the aggregationreducing mutations (7, 20) found in T1 (R2A, K5E, and N6D),
two are AB interface mutations (I125R and V127T), and four
are miscellaneous surface mutations (T21S, H41T, C117T, and
S131P).
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Construction of a Tandem Dimer of DsRed. Based on our early
results, it seemed as though engineering a true monomer of
DsRed might be impossible and therefore we pursued an alternate approach. The basic strategy was to fuse two copies of our
best AC dimer with a polypeptide linker such that the critical
dimer interactions could be satisfied through intramolecular
contacts with the tandem partner encoded within the same
polypeptide. Based on the crystal structure of the DsRed
tetramer (21, 22) a 10- to 20-residue linker could extend from the
C terminus of the A subunit to the N terminus of the C subunit
(⬇30 Å, Fig. 1B), but not to the N terminus of the B subunit (⬎70
Å). With our optimized dimer2 we constructed a series of
tandem constructs with linkers of varying lengths (9, 12, 13, or
22 aa) and a sequence similar to a known protease-resistant
Campbell et al.
linker (23). Of these four, only the tandem construct with the
nine-residue linker was notable for its somewhat slower maturation. The other three constructs were practically indistinguishable, and the tandem construct with the 12-residue linker,
designated tdimer2(12), is currently our preferred construct
because it has the shortest linker. As expected, dimer2 and
tdimer2(12) have identical excitation and emission maximum
and quantum yields (see Table 1). However, the extinction
coefficient of tdimer2(12) is twice that of dimer2 because of the
presence of two equally absorbing chromophores per polypeptide chain.
Evolution of a Monomer of DsRed. With the hope that our improved
dimers of DsRed would better tolerate disruption of the remaining interface, we constructed libraries in which AC interfacebreaking mutations were incorporated into our best dimers. Our
first such library targeted nine different positions, including two
key AC interface residues, H162 and A164, which were forced to
be either lysine or arginine. The brightest colonies from this
library were difficult to distinguish from the background red
fluorescence of the E. coli colonies even after prolonged imaging
with a digital camera. Suspect colonies were restreaked on
LB兾agar and allowed to mature at room temperature for 2
weeks, and a crude protein preparation was analyzed by SDS兾
PAGE. Imaging of the gel revealed a single faint band consistent
with the expected mass of the monomer and thus mRFP0.1 was
identified. Continuing with our directed evolution strategy for six
additional generations (see Tables 2 and 3 and primer lists)
resulted in drastic improvements in the brightness and rate of
maturation. The final clone, designated mRFP1, contains a total
of 33 mutations (Fig. 1C) relative to DsRed of which 13 are
internal to the -barrel (N42Q, V44A, V71A, K83L, F124L,
L150M, K163M, V175A, F177V, S179T, V195T, S197I, and
T217A). Of the 20 remaining external mutations, three are the
aggregation-reducing mutations from T1 (R2A, K5E, and N6D),
three are AB interface mutations (I125R, V127T, and I180T),
ten are AC interface mutations (R153E, H162K, A164R, L174D,
Y192A, Y194K, H222S, L223T, F224G, and L225A), and four
are additional beneficial mutations (T21S, H41T, C117E, and
V156A).
Characterization of dimer2, tdimer2(12), and mRFP1. Our initial
evidence for the monomeric structure of mRFP1 and its precursors was based on results with SDS兾PAGE (see Fig. 7, which
is published as supporting information on the PNAS web site)
and the lack of FRET between the green and red fluorescent
components in early generations. Thus analytical equilibrium
ultracentrifugation was performed on DsRed, dimer2, and
mRFP0.5a (an evolutionary precursor to mRFP1), and the
results confirmed the expected tetramer, dimer, and monomer
sizes (Fig. 2). DsRed, T1, and dimer2 (Fig. 3 A–C) all have a
fluorescent component that contributes at 475–486 nm to the
excitation spectra caused by FRET between oligomeric partners
(4). In T1 (Fig. 3B) this peak is quite pronounced, but in dimer2
PNAS 兩 June 11, 2002 兩 vol. 99 兩 no. 12 兩 7879
BIOCHEMISTRY
DsRed
T1
dimer2
tdimer2(12)
mRFP1
Rate of
photobleach
relative to EGFP
Fig. 4. Maturation of red fluorescence for DsRed, T1, dimer2, tdimer2(12),
and mRFP1. Log-phase cultures of E. coli expressing the construct of interest
were rapidly purified at 4°C, and beginning at 2 h postharvest their maturation at 37°C was monitored. The initial decrease in mRFP1 fluorescence is
attributed to a slight quenching on warming from 4°C to 37°C.
Fig. 2.
Analytical ultracentrifugation analysis of DsRed, dimer2, and
mRFP0.5a. The equilibrium radial absorbance profiles at 20,000 rpm were
modeled with a theoretical curve that allowed only the molecular weight to
vary. (A) The DsRed absorbance profile was best fit with an apparent molecular mass of 120 kDa, consistent with a tetramer. (B) The dimer2 absorbance
profile was best fit with an apparent mass weight of 60 kDa, consistent with
a dimer. (C) The mRFP0.5a absorbance profile was best fit with an apparent
molecular mass of 32 kDa, consistent with a monomer containing an Nterminal polyhistidine affinity tag.
(Fig. 3C), any excitation shoulder near 480 nm is almost obscured
by the 5-nm blue-shifted excitation peak. The 25-nm red-shifted
monomeric mRFP1 (Fig. 3D) also has a peak at 503 nm in the
absorption spectra, but in contrast to the other variants, this
species is nonfluorescent and therefore does not show up in the
excitation spectrum collected at any emission wavelength. The
green component of all DsRed variants may be caused by a
fraction of the protein that never matures beyond the green
intermediate (4, 24) or is trapped in a dead end product such as
a nonproductive trans conformation for the F65–Q66 peptide
bond (22). In either case it is not clear why this species would be
nonfluorescent in mRFP1. The large discrepancy in amplitude
between the 480-nm absorption and excitation peaks in T1
suggests that the corresponding species in T1 also has a very low
average quantum yield.
All red fluorescent proteins displayed double exponential
kinetics for photobleaching, suggesting a two-step process,
whereas EGFP under similar conditions bleached with very
nearly single exponential kinetics. Table 1 gives the rates of the
fast and slow components of the red proteins’ bleaching, normalized to the rate for EGFP bleaching with an equivalent
photon flux at EGFP’s absorbance maximum. Our bleaching
measurements of DsRed and EGFP are in good absolute
agreement with previous reports (for fuller discussion see additional Discussion and Table 4, which are published as supporting information on the PNAS web site).
As shown in Fig. 4, the rate of maturation of dimer2,
tdimer2(12), and mRFP1 is greatly accelerated over that of
DsRed although only mRFP1 matures at least as quickly as T1.
Based on our data at 37°C, we estimate the t0.5 for maturation of
mRFP1 and T1 is less than 1 h, consistent with the 0.7 h
previously reported for T1 (7). E. coli colonies expressing either
dimer2 or mRFP1 display similar or brighter levels of fluorescence to those expressing T1 after overnight incubation at 37°C
(see Fig. 8, which is published as supporting information on the
PNAS web site). The improved brightness of dimer2 may be
making up for its slower maturation. For mRFP1, which is less
bright than T1, we speculate that a higher folding efficiency is the
compensating factor.
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Expression of dimer2, tdimer2(12), and mRFP1 in Mammalian Cells.
Fig. 3. Fluorescence and absorption spectra of DsRed (A), T1 (B), dimer2 and
tdimer2(12) (C), and mRFP1 (D). The absorbance spectrum is shown with a solid
line, the excitation with a dotted line, and the emission with a dashed line.
7880 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.082243699
Although we suspected that we had evolved relatively robust red
fluorescent proteins, it was not known whether they were
suitable for expression in mammalian cells. Therefore dimer2,
tdimer2(12), and mRFP1 were expressed in transiently transfected HeLa cells. Within 12 h the cells displayed strong red
fluorescence evenly distributed throughout the nucleus and
cytoplasm (data not shown). This result prompted us to explore
a series of fusion constructs, using the gap junction protein Cx43,
which could demonstrate the advantage of a mRFP. A series of
constructs consisting of Cx43 fused to either GFP, T1, dimer2,
tdimer2(12), or mRFP1 were expressed in HeLa cells, which do
not express endogenous connexins. As previously reported (13)
the Cx43–GFP fusion protein was properly trafficked to the
membrane and was assembled into functional gap junctions,
whereas the Cx43–DsRed tetramer [T1 in this work, DsRed in
previous work (13)] consistently formed perinuclear localized
Campbell et al.
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Fig. 5. HeLa cells expressing Cx43 fused with T1, dimer2, or mRFP1. (A, C, and
E) Images were acquired with excitation at 568 nm (55 nm bandwidth) and
emission at 653 nm (95 nm bandwidth) with additional transmitted light.
Lucifer yellow fluorescence (B, D, and F) was acquired with excitation at 425
nm (45 nm bandpass) and emission at 535 nm (55 nm bandpass). (A) Two
contacting cells transfected with Cx43-mRFP1 and connected by a single large
gap junction. (B) One cell is microinjected with lucifer yellow at the point
indicated by * and the dye quickly passes (1–2 s) to the adjacent cell. (C) Four
neighboring cells transfected with Cx43-dimer2. The bright line between the
two rightmost cells is the result of having two fluorescent membranes in
contact and is not a gap junction. (D) As was observed about one-third of the
time, microinjected dye is slowly passed to an adjacent cell. (E) Two adjacent
cells transfected with Cx43-T1 and displaying the typical perinuclear localized
aggregation. (F) No dye passed between neighboring cells.
red fluorescent aggregates. Both Cx43–tdimer2(12) and Cx43–
dimer2 were properly trafficked to the membrane although
neither construct formed visible gap junctions. In contrast, the
Cx43–mRFP1 construct behaved identically to Cx43–GFP and
many red gap junctions were observed. Transfected cells were
microinjected with lucifer yellow to assess the functionality of the
gap junctions (see Fig. 5 and Table 5, which is published as
supporting information on the PNAS web site). The Cx43–
mRFP1 gap junctions rapidly and reliably passed dye, whereas
neither Cx43-T1-transfected cells nor nontransfected cells
passed dye. Interestingly, both Cx43– dimer2 and Cx43–
tdimer2(12) constructs slowly passed dye to a contacting transfected neighbor about one-third of the time. This apparently
‘‘gap junction independent’’ dye transfer may indicate that the
Campbell et al.
Discussion
Initially, we were faced with the decision of how to efficiently
construct and screen libraries of DsRed mutants such that we
could direct the evolution of tetrameric DsRed toward a mRFP.
Ultimately the best solution was to take the semirational approach of breaking the dimer interfaces in a stepwise fashion
(first AB then AC) and undertaking a directed evolution strategy
to rescue the red fluorescence. This combination of targeted and
random mutagenesis successfully directed the evolution of
DsRed from the poorly fluorescent dimer T1-I125R to the
monomeric mRFP1 in eight generations. In retrospect, breaking
up the tetramer was not the barrier to the discovery of mRFP1;
the challenge was to find the correct combination of many
mutations to rescue the red fluorescence in the crippled dimers
and monomers.
There are likely several mechanisms by which mutations in
dimer2 and mRFP1 have contributed to rescuing the red fluorescence. Although one mechanism probably involves improving
the folding efficiency or stability of the monomer, the most
important factor is undoubtedly the effect on the ratedetermining conversion from the ‘‘GFP-like’’ intermediate to
the DsRed chromophore (21, 24). Without crystal structures of
immature and mature forms of each mutant, little can be said
about the subtle effects each individual substitution may or may
not have on this poorly understood transformation. However, all
of the mutations discovered in this research and many of those
previously reported (4, 25) appear to cluster into three ‘‘hotspots.’’ The first of these hotspots is found in the plane of the
chromophore (when oriented as in Fig. 1C) and is defined by
N42, V44, and the Q66 side chain of the chromophore. The
mutations N42Q and V44A originated in T1 where they bestowed the tetramer with a greatly improved maturation time (7),
possibly by positioning Q66 in a conformation that promotes the
oxidation that transforms the green intermediate into the red
chromophore. Despite the extensive random and directed mutagenesis in this study, better mutations at or near these positions
were never identified, suggesting that these are nearly optimal
replacements. The second hotspot is just above the plane of the
chromophore, centered on the side chain K163 and influenced
by V175, F177, and possibly I161 (6). In the crystal structure of
DsRed, the phenolate anion of the chromophore is stabilized by
a salt bridge with the primary amine of K163 (22). Neither the
K163Q mutation in dimer2 nor the K163M mutation in mRFP1
can participate in a salt bridge and therefore the polarization of
the chromophore may be significantly different in these variants.
The third and most sensitive hotspot is found just below the plane
of the chromophore and is centered on the side chains of K70 and
the adjacent S197 and T217. Conservative mutations such as
K70R, S197T, and T217S had dramatic effects on the fluorescent
properties of DsRed mutants and were critical intermediate
steps toward mRFP1. Additionally, many of the beneficial
mutations in hydrophobic pockets of mRFP1, such as V71A and
L150M, are near this region and may be influencing the chromophore through subtle packing rearrangements that are mediated through this polar microenvironment. Interestingly, the
S197I mutation of mRFP1 is structurally analogous to the T203I
mutation in the sapphire variant of GFP, also known as H9–40
(2), where it is believed to destabilize the anionic form of the
chromophore. Considering that in mRFP1 the phenolate oxygen
is sandwiched between this same replacement and the nonpolar
K163M mutation, the overall effect should be to shift electron
density away from the phenolic anion. This repolarization may
PNAS 兩 June 11, 2002 兩 vol. 99 兩 no. 12 兩 7881
BIOCHEMISTRY
plasma membrane contains functional connexon channels that
are unable to assemble into gap junctions plaques because of
steric crowding of the fused dimer2 or tdimer2(12). Alternatively, dye transfer may be caused by gap junctions smaller than
the resolution of our camera.
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make the imidazolinone ring more electron rich and thereby
promote oxidation of the adjacent Q66 main-chain atoms to
form the acylimine of the extended DsRed chromophore (24).
The hydrophobic environment around the phenolic oxygen,
which is located in close proximity to the AC dimer interface,
may also serve to isolate and stabilize the critical hydrogen bond
to S146 and thus compensate for the loss of former AC interface
interactions. Fortunately, the pKa of the chromophore remains
low, ⬍5.0 (Table 1), despite these alterations in its immediate
environment.
The monomeric mRFP1 simultaneously overcomes the three
critical problems associated with the wild-type tetramer of
DsRed (4). Specifically mRFP1 is a monomer, it matures rapidly,
and it has minimal emission when excited at wavelengths optimal
for GFP. These features make mRFP1 the most suitable red
fluorescent protein for the construction of fusion proteins and
multicolor labeling in combination with GFP. As we have
demonstrated with the gap junction-forming protein Cx43,
mRFP1 fusion proteins are functional and trafficked identically
to their GFP analogues. Unfortunately, mRFP1 is not yet ideal
for all applications because its fluorescence quantum yield and
extinction coefficient (0.25 and 44,000 M⫺1䡠cm⫺1, respectively)
are significantly lower than for other DsRed variants (Table 1)
although much higher than HcRed1 (0.015 and 20,000
M⫺1䡠cm⫺1, respectively) (11). Also, mRFP1 photobleaches about
10-fold more easily than other DsRed variants, although its
photostability is still comparable to that for Aequorea EGFP
(26). Although the low extinction coefficient is attributable to
the significant fraction of the protein that remains trapped as a
nonfluorescent green species, the decreased fluorescence quantum yield and increased photobleaching quantum yield may
reflect imperfect shielding of the fluorophore by the surrounding cylindrical shell of -strands. The monomer has only one
layer of -strands separating the fluorophore from solvent and
oxygen, whereas the tetramer subunits should give each other
additional protection. Perhaps tight tetramerization of wild-type
coral proteins evolved to maximize thermotolerance or photostability under intense tropical sunlight; the progressive decrease
in oligomerization going from corals and corallimorphs (obligate
tetramer) to Renilla (southern U.S. waters, obligate dimer) and
Aequorea (Pacific Northwest, weak dimer) correlates with decreasing habitat temperature and intensity of daylight.
The extinction coefficient and fluorescence quantum yield
limit the brightness of fully mature mRFP1 to approximately
25% of DsRed, although for most imaging experiments this limit
will be more than compensated for by the greater than 10-fold
decrease in maturation time for mRFP1. However these factors
do render mRFP1 currently nonoptimal for the construction of
FRET-based sensors (unpublished observations). An interim
solution is to use the nonoligomerizing tdimer2(12), which is very
bright and displays FRET with all variants of Aequorea GFP
(unpublished observations). We expect that yet more desirable
mRFP variants with higher quantum yield and diminished green
component will be engineered. Similar multistep evolutionary
strategies, involving many rounds of evolution with few mutational steps per cycle, are likely to be profitable in converting
other oligomeric fluorescent proteins into monomers.
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7882 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.082243699
We thank Qing Xiong for preparation of various materials and Grant
Walkup, Stephen Adams, Jin Zhang, Alice Ting, and Brent Martin for
helpful discussion. We are grateful to Benjamin Glick for communicating
results in advance of publication and providing the T1 vector. R.E.C. is
supported in part by a postdoctoral fellowship from the Canadian Institutes
of Health Research. The work was supported by National Institutes of
Health Grants NS27177 (to R.Y.T.) and GM62114 (Alliance for Cell
Signaling), and the Howard Hughes Medical Institute. The DNA Sequencing Shared Resource at the University of California-San Diego Cancer
Center is funded in part by National Cancer Institute Cancer Support Grant
2P30CA23100-18.
Campbell et al.
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