Genetically encoded phenyl azide photochemistry drives positive and negative functional modulation of a red fluorescent protein

The mCherry gene (with a C-terminal hexahistidine tag) was cloned into the pBad/HisA vector (Invitrogen) using NcoI and XhoI restriction sites. Mutations were introduced by whole-plasmid inverse PCR using Phusion DNA polymerase (NEB) 1 and the primer pairs in Table S2. The introduced amber stop codon (TAG) mutations were incorporated in the forward primer. Mutations were confirmed by DNA sequencing.


Site-directed mutagenesis
The mCherry gene (with a C-terminal hexahistidine tag) was cloned into the pBad/HisA vector (Invitrogen) using NcoI and XhoI restriction sites.Mutations were introduced by whole-plasmid inverse PCR using Phusion ® DNA polymerase (NEB) 1 and the primer pairs in Table S2.The introduced amber stop codon (TAG) mutations were incorporated in the forward primer.Mutations were confirmed by DNA sequencing.

Protein purification
Proteins were purified by Ni-affinity chromatography using HisTrap TM HP column (GE Life Sciences) attached to an ÄKTApurifier (GE Life Sciences).Samples in PBS buffer (50 mM sodium phosphate, 100 mM NaCl, pH 8.0) were resolved at a flow rate of 1 mL/min.Protein elution was initiated by gradual increase in concentration of imidazole (0-250 mM) over 20 column volumes at a flow rate of 3 mL/min.Fractions containing mCherry protein were pooled and desalted in PBS buffer using Vivaspin 20 Sample Concentrator Unit 10,000 MWCO (GE Life Sciences).Proteins were further purified by size exclusion chromatography using Superdex 7510/300 GL column (GE Life Sciences) in PBS buffer at flow rate of 0.5 mL/min.Eluted fractions containing native or mutant mCherry were pooled were pooled and collected to a final volume of 1 mL.Elution was monitored by absorption at 280 nm (general protein absorbance) and 587 nm (mCherry specific absorbance).Protein purity was analysed by SDS-PAGE.

Fluorescence and Absorbance Spectroscopy
Fluorescence.Fluorescence excitation and emission spectra measured in a 5 × 5 mm QS quartz cuvette (Hellma).Excitation and emission spectra were recorded at 20°C and scan rate of 600 nm/min with a slit width of 10 nm (5 nm for wild-type mCherry) using Cary Eclipse fluorescence spectrophotometer (Varian).UV irradiation was performed in the QS cuvette using a 6 W UVM-57 mid-range UV handheld lamp (UVP) with a peak emission at 310 nm with the lamp at a distance of 1 cm from the sample.Live cell and cell lysate samples containing mCherry azF variants were diluted with PBS (100 mM sodium phosphate, 300 mM NaCl, pH 8) in a 1:1 volume ratio.Wild-type mCherry samples were diluted with PBS in a 1:6 ratio.Purified proteins were measured at a concentration of 1 µM in PBS.Cell lysates containing PAmCHerry 197Y , PEmCherry 67amF or PEmCherry 67Y were diluted at 1:2 ratio in PBS and placed in a 5 × 5 mm QS quartz cuvette.Quantum yields for each mCherry variant were calculated using wild-type mCherry as a reference (known φ = 0.22).Pure protein sample were diluted to a final absorbance of 0.05 at their respective λ max and fluorescence emission spectra were recorded.Integrated emission intensity was calculated and used in the following formula to generate quantum yield values where the φx and φst refer to the fluorescence quantum yield of the sample and fluorescein standard, respectively.Areax and Areast are the integrated emission intensities for the sample and fluorescein standard, respectively.ηx and ηst is the refractive index of the solvent for the sample and fluorescein standard, respectively.The refractive index correction here was negligible as 0.1 M NaOH and aqueous buffers differ in refractive index by <1% 1 .
Absorbance.Purified proteins samples were diluted in PBS buffer to a final concentration of 20µM and placed in a 10 × 10 mm QS quartz cuvette.UV-vis absorbance spectra were recorded at 20°C using HP 8452A Diode Array Spectrophotometer (Hewlett Packard).Protein irradiation was performed as described above.Brightness was calculated by multiplying ε by quantum yield.

Widefield fluorescence microscopy
Widefield fluorescence microscopy was performed essentially as described previously 1 .E. coli TOP 10 cells producing PDmCherry W143azF or PAmCherry I197azF were immobilised within 2% low melting point agarose and mounted onto microscope slides.Samples were imaged using an Olympus IX73 inverted microscope utilising the ET-Sedat quad filter set (#89000 from Chroma), coupled to an ORCA Flash 4.0 camera (Hamamatsu).Photoactivation and photodeactivation experiments were performed using a Procan220 (Prior) 200W metal halide light source transmitted through a ET555/25x excitation filter for 500 ms, followed by irradiation through an AT350/50x excitation filter for 30 ms.Each imaging cycle (of visualisation and irradiation) took 2 s.Photocontrol experiments were performed using the same cycle of visualisation and irradiation.The cells were visualised using 100x oil immersion objective at 4x binning and imaged utilising the HCImage Live (Hamamatsu) software.Data was analysed with ImageJ software 3 .

X-ray Crystallography
PDmCherry 143azF was produced and purified in the dark and diluted in 50 mM Tris-HCl (pH 8) to 10 mg/mL.Crystal screens were set up using the sitting drop vapour diffusion method with incubation at 4 °C.The screening conditions were based on 50 mM Tris-HCl with varying pH (7.9 -8.9) and PEG4000 concentration (25 -32%).Drops were set up with 0.2 µL of protein and reservoir solutions and dispensed with an Art Robins Phoenix robot (AlphaBiotech, UK).Crystals were transferred to mother liquor supplemented with 13% (w/v) ethylene glycol as a cryoprotectant and vitrified.Data were collected at beamline I02 (dark) and I03 (irradiated) of the Diamond Light Source (Harwell, UK).Data were reduced with the XIA2 package 4 , space group assignment by POINTLESS 5 , scaling and merging were completed with AIMLESS 6 and TRUNCATE 7 .Structures were solved by molecular replacement with the structure of mCherry (PDB 2H5Q) using PHASER 8 .Structures were manually adjusted to the electron density using COOT 9 and refined by TLS restrained refinement using RefMac 10 .All non-protein atoms were refined isotropically.The above routines were used as the CCP4 package (www.ccp4.ac.uk).Graphical representations were made with PyMOL Molecular Graphics System, Schrödinger, LLC.
Crystals of the dark state of PDmCherry 143azF formed at 27-28% PEG4000, pH 7.9-8.3,and the best crystal diffracted to 1.7 Å.The irradiated state of PDmCherry 143azF was obtained by irradiating the crystal tray containing remnant dark state PDmCherry 143azF crystals for 1 hour.The best data for the irradiated state of PDmCherry 143azF diffracted to 2.0 Å.Both the dark and irradiated forms of PDmCherry 143azF crystallized in the orthorhombic space group P2(1)2(1)2(1) and contained three molecules in the asymmetric unit.Final refinement statistics and model geometry are summarized in Table S3.In close proximity to Tyr-moiety of CRO.

Q163
Residue was mutated from M in mRFP1.4 to produce mCherry.

I197
In close proximity to Tyr-moiety of CRO; lies above plane close to K70.I197E substitution in mTangerine produced mBanana [
Residue was mutated to M from original Q in mRFP1 to produce mCherry.Residue mutated to W to produce mOrange and mStrawberry[1]   Y67Within CRO (comprises phenol moiety).Residue mutated to W to produce mHoneydew[1] Equivalent to sfGFP Y66azF [2] K70 Close to Gly-moiety of CRO.K70N was a key substitution in mCherry to generate PAmCherry [3] W93 Close to Gly-moiety of CRO.Y120 Close to Met and Gly moieties of CRO W143 Tryptophan has a similar size to AzF.Could take conformation pointing towards Tyr-moiety of CRO.Equivalent to sfGFP F145azF [2] S146 Directly interacts with CRO phenol OH group.Equivalent to sfGFP H148azF [2] E148 Side chain points toward CRO and lies approximately parallel to CRO plane.Important structural residue.I161

Fig S2 .
Fig S2.Response of wild type mCherry to irradiation.(a) Change in fluorescence emission (excitation at 585 nm) after 1 hour UV irradiation.(b) Change in emission under cell imaging conditions.Cells were imaged at 555 nm (AlexaA) for 500 ms, followed by irradiation at 350 nm (DAPI) for 30 ms, cycled every 2 seconds.

Fig S4 .Fig S5 .Fig S7 .
Fig S4.Structural comparison between mCherry (PDB H5Q; magenta) and superfolder GFP (PDB 2B3P; green) of the chromophore (CRO) and the residues equivalent to W143 in mCherry.A and B show 2 different views rotated by about 45° about the x axis.

Table S1 .
Properties of azF-containing mCherry variants.NF refers to no observable fluorescence; NE no observable expression.
1] L199Close to M and Y moieties of CRO.