Extended Stokes Shift in Fluorescent Proteins: ChromophoreProtein Interactions in a Near-Infrared TagRFP675 Variant
Kiryl D. Piatkevich, Vladimir N. Malashkevich, Kateryna S. Morozova, Nicolai A. Nemkovich,
Steven C. Almo, and Vladislav V. Verkhusha
Supporting Information
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EGFP
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATY-GKLTLKFICTT-GKLPVPWPTL
mPlum
MVSKGEENNMAIIKEFMRFKEHMEGSVNGHEFEIEGEGEGRP-YEGTQTARLKVTKGGPLPFAWDIL
mRouge
MVSKGEEDNMAIIKEFMRFKTHMEGSVNGHEFEIEGEGEGRP-YEGTQTAKLKVTKGGPLPFAWDIL
E2-Crimson
MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKP-YEGTQTAKLQVTKGGPLPFAWDIL
mKate
MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKP-YEGTQTMRIKVVEGGPLPFAFDIL
GmKate
MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKP-YEGTQTMRIKVVEGGPLPFAFDIL
mNeptune
MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKP-YEGTQTGRIKVVEGGPLPFAFDIL
eqFP650
MGEDSELISENMHMKLYMEGTVNGHHFKCTSEGEGKP-YEGTQTAKIKVVEGGPLPFAFDIL
eqFP670
MGEDSELISENMHTKLYMEGTVNGHHFKCTSEGEGKP-YEGTQTCKIKVVEGGPLPFAFDIL
TagRFP657
MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKP-YEGTQTQRIKVVEGGPLPFAFDIL
TagRFP675
MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKP-YEGTQTQRIKVVEGGPLPFAFDIL
EGFP
mPlum
mRouge
E2-Crimson
mKate
GmKate
mNeptune
eqFP650
eqFP670
TagRFP657
TagRFP675
70
80
90
100
110
120
VTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV
SPQIQYGSKAYVKHPADIP--DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI
SPQFQYGSKAYVKHPADIP--DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI
SPQFFYGSKAYIKHPADIP--DYLKQSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGTLI
ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
ATCFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGCLI
ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGCLI
ATSFMYGSHTFINHTQGIP--DFWKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
ATSFMYGSKTFINHTQGIP--DFWKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
EGFP
mPlum
mRouge
E2-Crimson
mKate
GmKate
mNeptune
eqFP650
eqFP670
TagRFP657
TagRFP675
- 130
140
150
160
170
180
NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD
YKVKVRGTNFPSDGPVMQKKTMG-WEASTERMYPE--DGALKGEMKMRLRLKDGGHYDAE
YKVKLRGTNFPSDGPVMQKKTMG-WEACSERMYPE--DGALKGEMKMRLKLKDGGHYDAE
YHVKFIGVNFPSDGPVMQKKTLG-WEPSTERNYPR--DGVLKGENHMALKLKGGGHYLCE
YNVKIRGVNFPSNGPVMQKKTLG-WEASTEMLYPA--DGGLEGRSDMALKLVGGGHLICN
YNVKIRGVNFPSNGPVMQKKTLG-WEACTEMLYPA--DGGLEGRADMALKLVGGGHLICN
YNVKIRGVNFPSNGPVMQKKTLG-WEASTETLYPA--DGGLEGRCDMALKLVGGGHLICN
YNVKINGVNFPSNGPVMQKKTLG-WEASTEMLYPA--DSGLRGHSQMALKLVGGGYLHCS
YNVKINGVNFPSNGPVMQKKTLG-WEANTEMLYPA--DSGLRGHNQMALKLVGGGYLHCS
YNVKIRGVNFPSNGPVMQKKTLG-WEAHTEMLYPA--DGGLEGRTALALKLVGGGHLICN
YNVKIRGVNFPSNGPVMQKKTLG-WEANTEMMYPA--DGGLEGRNYMALKLVGGGHLICS
EGFP
mPlum
mRouge
E2-Crimson
mKate
GmKate
mNeptune
eqFP650
eqFP670
TagRFP657
TagRFP675
190
200
210
220
230
HYQQNTPIGD-GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
VKTTYMAKKP---VQLPGAYKTDIKLDITSH-NEDYTIVEQYERAEGRHSTGGMDELYK
VKTTYKAKKP---VQLPGAYNTNTKLDITSH-NEDYTIVEQYERNEGRHSTGGMDELYK
FKSIYMAKKP---VKLPGYHYVDYKLDITSH-NEDYTVVEQYERAEARHHLFQ
LKTTYRSKKPAKNLKMPGVYYVDRRLERIKE-ADKETYVEQHEVAVARYCDLPSKLGHK
LKTTYRSKKPAKNLKMPGVYYVDRRLERIKE-ADKETYVEQHEVAVARYCDLPSKLGHK
LKTTYRSKKPAKNLKMPGVYFVDRRLERIKE-ADNETYVEQHEVAVARYCDLPSKLGHKLN
LKTTYRSKKPAKNLKMPGFYFVDRKLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS
LKTTYRSKKPAKNLKMPGFYFVDRKLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS
FKTTYRSKKPAKNLKMPGVYYVDYRLERIKE-ADKETYVEQHEVAVARYCDLPSKLGHKLN
LKTTYRSKKPAKNLKMPGVYYVDRRLERIKE-ADKETYVEQHEVAVARYCDLPSKLGHKLN
Supporting Figure 1. Alignment of the amino acid sequences for TagRFP675, far-red FPs, and
EGFP. Internal amino acids are highlighted in gray. The chromophore-forming residues are
underlined. Mutations resulting in the conversion of mKate into TagRFP675 are shown in red.
The residues highlighted in green are suggested being responsible for the bathochromic shift of
emission in the respective FPs. The alignment numbering follows that for EGFP.
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Supporting Figure 2. Molecular structure of the chromophore superimposed onto the refined
2Fo-Fc electron density. (A) A subunit, pH 8.0. The chromophore adopts predominantly cisconfiguration. (B) B subunit B, pH 8.0. The chromophore adopts cis-configuration (occupancy
0.6) and trans-configuration (occupancy 0.4). (C) A subunit, pH 4.5. The chromophore adopts
predominantly cis-configuration. (D) B subunit, pH 4.5. The chromophore adopts cisconfiguration (occupancy 0.5) and trans-configuration (occupancy 0.5). The occupancy of cisand trans-chromophore was calculated during structure refinement.
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A
B
Supporting Figure 3. Emission spectra of TagRFP675 at pH 4.5. (A) Emission spectrum at 460
nm excitation. (B) Emission spectrum at 570 nm excitation.
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A
B
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C
D
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E
F
Supporting Figure 4. Instantaneous fluorescence spectra of (A) TagRFP675, (B)
TagRFP675/Q41M, (C) TagRFP675/Q41P, (D) TagRFP675/N143S, (E) TagRFP675/N158K,
and (F) mKate measured at different registration times, indicated at each plot. The measurements
were performed at 298 K with excitation wavelength 337.1 nm. The arrows indicate maximum of
fluorescence emission, λ, wavelength, and τ, fluorescence life-time.
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A
C
B
D
Supporting Figure 5. Emission spectra of TagRFP675, TagRFP675/Q41M, TagRFP675/Q41P
and mKate at different temperatures. (A) Emission of TagRFP765 at 298K (red line), 196K
(cyan line) and 77K (blue line). (B) Emission of TagRFP765/Q41M at 298K (red line), 196K
(cyan line) and 77K (blue line). (C) Emission of TagRFP765/Q41P at 298K (red line), 196K
(cyan line) and 77K (blue line). (D) Emission of mKate at 298K (red line), 196K (cyan line) and
77K (blue line).
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Supporting Figure 6. Emission spectra of TagRFP675 at various excitation wavelengths at
room temperature.
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Supporting Table 1. Data collection and refinement statistics.
TagRFP675 (pH 4.5)
TagRFP675 (pH 8.0)
Beamline
NSLS-X29A
NSLS-X29A
Wavelength (Å)
1.08
1.08
Resolution limits (Å)
20–2.30
20–2.30
Observed reflections
692565
557113
Unique reflections
32982
35919
Completeness (%)
99.5 (100.0)a
99.2 (100.0)a
Rmergeb
0.090 (0.660)a
0.074 (0.740)a
Protein non-hydrogen atoms
3643
3604
Water molecules
243
169
Rcrystc
0.188 (0.208)a
0.191 (0.189)a
Rfreec
0.232 (0.259)a
0.233 (0.261)a
Average B-factor (Å2)
40.5
34.2
Bond length, Å
0.008
0.009
Bond angles (°)
1.55
1.66
Torsion angles (°)
24.2
25.6
Core (%)
98.1
98.1
Allowed (%)
1.6
1.6
Generous (%)
0.3
0.3
Data collection
Refinement statistics
RMSD from ideality
Ramachandran plot
Values in parentheses indicate statistics for the high resolution bin. b Rmerge = j|Ij(hkl) –
<I(hkl)>|/ j|<I(hkl)>|, where Ij is the intensity measurement for reflection j and <I> is the
mean intensity over j reflections. c Rcryst/(Rfree) =  ||Fo(hkl)| – |Fc(hkl)||/ |Fo(hkl)|, where Fo and
Fc are observed and calculated structure factors, respectively. No -cutoff was applied. 5% of
the reflections were excluded from refinement and used to calculate Rfree.
a
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