Extended Stokes Shift in Fluorescent Proteins: Chromophore-Protein 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
10 20 30 40 50 60
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
70 80 90 100 110 120
EGFP vttfTygvqcfsrypdhmkqhdffksampegyvqertiffkddgnyktraevkfeGDtLV
mPlum SPQIQYGSKAYVKHPADIP--DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI
mRouge SPQFQYGSKAYVKHPADIP--DYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI
E2-Crimson SPQFFYGSKAYIKHPADIP--DYLKQSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGTLI
mKate ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
GmKate ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
mNeptune ATCFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
eqFP650 ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGCLI
eqFP670 ATSFMYGSKTFINHTQGIP--DFFKQSFPEGFTWERITTYEDGGVLTATQDTSLQNGCLI
TagRFP657 ATSFMYGSHTFINHTQGIP--DFWKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
TagRFP675 ATSFMYGSKTFINHTQGIP--DFWKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLI
- 130 140 150 160 170 180
EGFP nrielkgidfkedgnilghkleynynshnvyimadkqkngikvnfkirhniedgSVQLad
mPlum YKVKVRGTNFPSDGPVMQKKTMG-WEASTERMYPE--DGALKGEMKMRLRLKDGGHYDAE
mRouge YKVKLRGTNFPSDGPVMQKKTMG-WEACSERMYPE--DGALKGEMKMRLKLKDGGHYDAE
E2-Crimson YHVKFIGVNFPSDGPVMQKKTLG-WEPSTERNYPR--DGVLKGENHMALKLKGGGHYLCE
mKate YNVKIRGVNFPSNGPVMQKKTLG-WEASTEMLYPA--DGGLEGRSDMALKLVGGGHLICN
GmKate YNVKIRGVNFPSNGPVMQKKTLG-WEACTEMLYPA--DGGLEGRADMALKLVGGGHLICN
mNeptune YNVKIRGVNFPSNGPVMQKKTLG-WEASTETLYPA--DGGLEGRCDMALKLVGGGHLICN
eqFP650 YNVKINGVNFPSNGPVMQKKTLG-WEASTEMLYPA--DSGLRGHSQMALKLVGGGYLHCS
eqFP670 YNVKINGVNFPSNGPVMQKKTLG-WEANTEMLYPA--DSGLRGHNQMALKLVGGGYLHCS
TagRFP657 YNVKIRGVNFPSNGPVMQKKTLG-WEAHTEMLYPA--DGGLEGRTALALKLVGGGHLICN
TagRFP675 YNVKIRGVNFPSNGPVMQKKTLG-WEANTEMMYPA--DGGLEGRNYMALKLVGGGHLICS
190 200 210 220 230
EGFP hyqqntpigd-gpvllpdnhylstqsalskdpnekrdhmvllefvtaagithgmdelyk
mPlum VKTTYMAKKP---VQLPGAYKTDIKLDITSH-NEDYTIVEQYERAEGRHSTggmdelyk
mRouge VKTTYKAKKP---VQLPGAYNTNTKLDITSH-NEDYTIVEQYERNEGRHSTGGMDELYK
E2-Crimson FKSIYMAKKP---VKLPGYHYVDYKLDITSH-NEDYTVVEQYERAEARHHLFQ
mKate LKTTYRSKKPAKNLKMPGVYYVDRRLERIKE-ADKETYVEQHEVAVARYCDLPSKLGHK
GmKate LKTTYRSKKPAKNLKMPGVYYVDRRLERIKE-ADKETYVEQHEVAVARYCDLPSKLGHK
mNeptune LKTTYRSKKPAKNLKMPGVYFVDRRLERIKE-ADNETYVEQHEVAVARYCDLPSKLGHKLN
eqFP650 LKTTYRSKKPAKNLKMPGFYFVDRKLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS
eqFP670 LKTTYRSKKPAKNLKMPGFYFVDRKLERIKE-ADKETYVEQHEMAVARYCDLPSKLGHS
TagRFP657 FKTTYRSKKPAKNLKMPGVYYVDYRLERIKE-ADKETYVEQHEVAVARYCDLPSKLGHKLN
TagRFP675 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.
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 cis-configuration. (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 cis-configuration (occupancy 0.5) and trans-configuration (occupancy 0.5). The occupancy of cis- and trans-chromophore was calculated during structure refinement.
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.
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.
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).
Supporting Figure 6. Emission spectra of TagRFP675 at various excitation wavelengths at room temperature.
Supporting Table 1. Data collection and refinement statistics.
TagRFP675 (pH 4.5) TagRFP675 (pH 8.0)
Data collection
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
Refinement statistics
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
RMSD from ideality
Bond length, Å 0.008 0.009
Bond angles (°) 1.55 1.66
Torsion angles (°) 24.2 25.6
Ramachandran plot
Core (%) 98.1 98.1
Allowed (%) 1.6 1.6
Generous (%) 0.3 0.3
a Values in parentheses indicate statistics for the high resolution bin. b Rmerge = SS j|Ij(hkl) – <I(hkl)>|/ SS j|<I(hkl)>|, where Ij is the intensity measurement for reflection j and <I> is the mean intensity over j reflections. c Rcryst/(Rfree) = S ||Fo(hkl)| – |Fc(hkl)||/ S |Fo(hkl)|, where Fo and Fc are observed and calculated structure factors, respectively. No s-cutoff was applied. 5% of the reflections were excluded from refinement and used to calculate Rfree.
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