Photoactivatable Blue Fluorescent Protein

Photoactivatable and photoswitchable fluorescent proteins (FPs) are sophisticated molecular tools that in combination with super-resolution microscopy are helping to elucidate many biological processes. Through the Y66H mutation in the chromophore of the violet fluorescent protein SumireF, we created the first photoactivatable blue fluorescent protein (PA-BFP). This protein is rapidly activated over ordinary UV transilluminators at 302 or 365 nm in irreversible mode and by direct exposition to sunlight. The maximum excitation and emission wavelengths of this protein, centered at 358 and 445 nm, respectively, resemble the values of DAPI—the blue stain widely used in fluorescence microscopy to visualize nucleic acids in cells. Therefore, the immediate use of PA-BFP in cellular biology is clear because the technology required to follow this new genetically encoded reporter at the microscopic level has already been established. PA-BFP can potentially be used together with other photoactivatable fluorescent proteins of different colors to label multiple proteins, which can be simultaneously tracked by advanced microscopic techniques.


■ INTRODUCTION
−6 The ocean contains thousands of fluorescent and chemiluminescent organisms whose fluorescent proteins or luciferases await discovery. 7,8−11 Here, we describe the discovery and characterization of an interesting photoactivatable blue fluorescent protein (PA-BFP) derived from the recently published violet protein Sumire. 12

■ RESULTS AND DISCUSSION
The violet fluorescent protein Sumire contains a green fluorescent protein (GFP)-type chromophore whose imidazolidinone ring remains hydroxylated, shortening the delocalization of π orbitals as compared to the ordinary GFP chromophore (Figure S1). 12−14 Consequently, Sumire displays blue-shifted excitation and emission wavelengths to 340 and 414 nm, respectively, taking the original GFP as a reference, whose maximum excitation and emission wavelengths are centered at 395 and 509 nm, respectively. 15umire was derived from superfolder GFP (sfGFP) 16 through nine site-directed mutations: T65G, Q69A, Y145G, N146I, H148G, F165Y, T203V, S205V, and V224R. 12owever, when we assembled the gene, as described in the Methods section, following a procedure modified from the original, a fortuitous variant became violet at a faster rate than the target Sumire protein (Figure S2).This improved variant contained the original phenylalanine amino acid in position 165 instead of the expected tyrosine residue and was named SumireF.
Directed evolution of the SumireF gene by error-prone PCR mutagenesis 17 and screening of the library over a UV transilluminator at 302 nm revealed the presence of a colony displaying a brilliant sky-blue phenotype (Figure 1A).DNA sequencing of the plasmid recovered from this colony confirmed that the encoded protein contained the mutation Y66H in the center of the chromophore, as inferred by a sequence comparison with the parental proteins Sumire, SumireF, and sfGFP (Figure 1B).−21 However, when E. coli cells were transformed with the plasmid harboring the mutant gene SumireF-Y66H, the resultant colonies required 2 min over the 302 or 365 nm UV transilluminator to fluoresce blue, as shown in the accompanying videos (see PA-BFP activation at 302 nm speed 4X.mp4 and PA-BFP activation at 365 nm speed 4X.mp4).Photoactivation was also confirmed by fluorescence spectroscopy.When E. coli cells producing the "dark" SumireF-Y66H heterologous protein were scraped from a Petri dish, resuspended in phosphate buffer saline (PBS), and subjected to repetitive acquisition of emission spectra every 2 min, with excitation at 365 nm, the fluorescence grew in each cycle due to the cumulative fraction of the activated protein, as shown in Figure 2A.This experiment repeated 45 days later with cells stored at 4 °C in the refrigerator produced the same result as with fresh cells, demonstrating that the dark state of the protein is very stable, as shown in Figure S3A.Even more, the dark state is also stable at the benchtop after hours of ordinary illumination in the laboratory; however, it becomes fully activated within 3 min when exposed to direct sunlight due to its UV content, as shown in Figure S3B,C, respectively.
Kinetic activation of the pure dark protein, followed by fluorescence spectroscopy, demonstrated that activation at 302 nm was faster than activation at 365 nm, as shown in Figure 2B.At 302 nm, the activation half-life of the protein was 20.6 min, whereas at 365 nm, it was 47.0 min.The activation followed a first-order kinetic reaction.Complete activation of the protein, either pure or expressed in cells, is much faster on the UV transilluminator than on the fluorescence spectrometer because of the size of the excitation window (i.e., the entire screen on the transilluminator versus only 15 nm, which is the maximum excitation slit allowed in the fluorescence spectrometer at our disposal).
From this point, the protein was named PA-BFP.There are many photoactivatable fluorescent proteins of different colors, including cyan, 22 green, 23 yellow, 24 orange, 25 and red, 26,27 but PA-BFP seems to be the first photoactivatable protein in the blue range.Purification of the heterologous protein by immobilized nickel affinity chromatography yielded the "dark" protein mentioned above, whose maximum absorption was centered at 329 nm (Figure 3A).Excitation at this wavelength produced an emission peak at 397 nm in the violet region of the visible spectrum (Figure 3B).These blue-shifted values, compared to the parental protein, suggest a Sumire-like intermediate structure in which the phenol moiety of the chromophore is replaced by an imidazole ring, and the cyclized imidazolidinone ring remains in the hydroxylated form, as proposed in the post-translational mechanism shown in Figure 4.The exposition of the intermediate protein at 365 nm over a UV transilluminator for 3 min produced a new protein version whose maximum absorbance was centered at 360 nm (Figure 3A).Excitation at this wavelength produced an emission peak at 445 nm, as shown in Figure 3B.Contrary to the PA-BFP, the exposition of SumireF to UV did not produce any phenotypical changes to the protein.Originally, the Sumire protein was partially designed from the photoswitchable yellow fluorescent protein (YFP) Dreiklang�a protein whose chromophore remains in the hydroxylated form and is activated with UV light at 365 nm to produce YFP and reverts to the dark state by irradiation with violet light at 405 nm. 28PA-BFP activation was irreversible, and exposing the protein to violet light did not produce a return to the dark state.Therefore, we hypothesize that PA-BFP is activated via a photochemical dehydroxylation process as the last step in the maturation pathway of the chromophore (Figure 4) because fluorescence emerges immediately after UV exposure.Thus, the cyclization and oxidation steps must precede dehydroxylation because the dark intermediate is already oxidized; it contains two fewer hydrogens than the expected unmodified polypeptide, as confirmed by electrospray ionization-mass spectrometry-liquid chromatography (ESI-MS-LC).The expected molecular weight of the unmodified polypeptide is 27,544 Da, whereas the experimental value of the dark intermediate is 27,541.98Da (Figure S4A), which has two fewer hydrogens than the complete polypeptide due to dehydrogenation of the C α −C β bond of histidine 66.After UV activation, the dark intermediate lost 19 Da, whereas we expected only 18 Da, corresponding to the elimination of a water molecule (Figure S4B).Therefore, as suggested in the last part of the proposed mechanism, one additional proton is probably lost by ionization of the chromophore.Ultimately, the chromophore of PA-BFP (Figure 4) must be the same as that present in classical BFPs derived from the original Aequorea victoria GFP.Structural analysis of PA-BFP and site-directed mutagenesis will likely identify those amino acids important for arresting protein maturation in the oxidation step.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of PA-BFP revealed the presence of only one band around 28 kDa after boiling the sample under denaturing conditions (Figure S5).This ruled out breakage of the polypeptide backbone, as occurs in the activation of kaede, 29 EosFP, 30 KiKGR, 31 and Dendra 32 and further supported photochemical dehydroxylation as the final step in the activation of the protein.
Finally, Figure 1A clearly reveals that PA-BFP is more fluorescent than its parent protein SumireF, a qualitative result that was quantitatively confirmed by determining the optical properties of both proteins.PA-BFP exhibited a molar extinction coefficient of 13983 M −1 cm −1 and a quantum yield of 0.516, whereas the results for SumireF were 23065 M −1 cm −1 and 0.191, respectively.The brightness of a protein is the product of both values divided by 1000.Therefore, the brightness of PA-BFP is 7.2 versus 4.4 for SumireF.PA-BFP was found in the first round of random mutagenesis, perhaps more mutation rounds combined with selection by fluorescence-activated cell sorting will improve its maturation efficiency and, consequently, its molar extinction coefficient to yield a more fluorescent PA-BFP as has been achieved with other fluorescent proteins. 33he fluorescent properties of PA-BFP resemble those exhibited by DAPI (Ex max : 358 nm, Em max : 461 nm)�the universal dye employed to stain nucleic acids in cells.Most fluorescence microscopes are equipped with filters for DAPI; thus, the technology has already been established for use with this new protein reporter.
In summary, we have added a new tool to the palette of photoactivated fluorescent proteins that has the appropriate properties to be used in multiple-labeling experiments and analyzed by photoactivated localization microscopy 34 and other super-resolution microscopy techniques. 35Furthermore, size exclusion chromatography of PA-BFP demonstrated that this protein is a monomer like its parental precursors sfGFP and SumireF (Figure S6); therefore, it can be used to label other proteins of biological interest without the risk of forming insoluble aggregates.
■ METHODS Sumire Gene Construction.The Sumire gene reported by Sugiura and Nagai 12 was assembled by site-directed mutagenesis using the sfGFP 16 gene as a template and three pairs of oligonucleotides that grouped the expected mutations: T65G/Q69A, Y145G/N146I/H148G/F165Y, and T203V/ S205V/V224R.These changes were incorporated into the sfGFP gene one at a time using the overlapped PCR approach. 36The gene was cloned as a NdeI/BamHI insert into our constitutive pJOQ plasmid, which was previously described. 37For purification purposes, any insert cloned with this pair of restriction sites encodes a polyhistidine tag at the carboxy end of the target protein.The ligation reaction was used to transform the electrocompetent cells of E. coli MC1061.The correct assembly of the Sumire gene was confirmed by DNA sequencing of some of the transformants that displayed a slightly violet fluorescent phenotype when the Petri dishes were analyzed over a UV transilluminator at 302 nm after incubation at 37 °C for 20 h and storage at 4 °C for 24 h.In this process of sequence confirmation, a fortuitously improved Sumire mutant was discovered.It contained all the expected mutations except F165Y.This mutant, which was more fluorescent than Sumire and did not require incubation at 4 °C to complete the process of chromophore maturation, was named SumireF.
Generation of Photoactivatable Blue Fluorescent Protein.The SumireF gene was subjected to random PCR diversification using the GeneMorph II mutagenesis kit under the conditions suggested by the manufacturer to produce 3−4 nucleotide changes per 1 Kb of the extended template. 17The library of mutant genes was cloned and transformed into E. coli cells, as described for the Sumire gene.We found a brilliant sky-blue variant after screening approximately 20,000 colonies over a UV transilluminator at 302 nm.This mutant contained the mutation Y66H localized in the center of the chromophore.E. coli cells were electroporated with the plasmid Sumire-Y66H and grown on solid LB/kanamycin at 37 °C for 20 h.Visualization of the Petri dishes over a UV transilluminator at 302 or 365 nm (benchtop 2UV transilluminator from UVP supplemented with 8W 302 and 365 nm UV lamps) converted the nonfluorescent colonies into intense sky-blue brilliant colonies after 2 min of exposition.The new protein was named PA-BFP.
Protein Purification.To begin protein purification, 250 mL of Luria−Bertani broth containing kanamycin at 30 μg/mL was inoculated with a colony of PA-BFP, and the culture was incubated at 37 °C for 24 h under agitation at 200 rpm.Next, the cells were recovered by centrifugation and resuspended in 12 mL of PBS buffer.The suspension was cooled in ice water and sonicated twice for 2 min with intermediate cooling.The cell debris was removed by centrifugation, and the supernatant was loaded onto a purification column containing 5 g of Ni-NTA resin, which was previously equilibrated with 0.5 M NaCl in 0.1 M phosphate buffer (pH 7.0, buffer A).The stationary phase was washed with 15 mL of the same buffer, followed by 15 mL of 30 mM of imidazole in buffer A. The heterologous protein was then eluted with 15 mL of 0.3 M imidazole in buffer A. Finally, the protein was concentrated in a centrifugal filter (Amicon Ultra-15, 10K, Millipore) and washed twice with 15 mL of PBS to remove the imidazole.The protein was recovered with 1 mL of PBS buffer, and a small fraction was saved at 4 °C as a "dark" species for subsequent experiments and analyses.The remaining protein was transferred to a quartz cuvette and activated for 3 min at 365 nm over a UV transilluminator.The parental protein SumireF was purified in the same way.Both proteins�SumireF and activated PA-BFP�were analyzed by SDS-PAGE electrophoresis, revealing the presence of only one band of the expected size (28 kDa) when the sample was boiled and two bands when the sample was analyzed under semidenaturing conditions.One of these bands corresponded to the denatured protein, whereas the other corresponded to the native, undenatured protein.
UV−Visible Spectroscopy.The UV−visible (UV−vis) spectra of the pure protein PA-BFP in its dark and activated state dissolved in PBS, as well as SumireF, were obtained by recording their absorbance in the range 250−550 nm using silica quartz cuvettes on the spectrophotometer IMPLEM.Fluorescence Characterization.The excitation and emission spectra of the pure proteins diluted in PBS were recorded on an LS-55 fluorescence spectrometer from PerkinElmer.The emission spectrum of each protein was obtained by fixing the excitation wavelength at its maximum absorbance wavelength, determined by UV−vis absorbance, and recording the emission in the range "excitation wavelength +10 nm" up to 700 nm using 5 nm emission and excitation slits.The excitation spectra were recorded in a similar mode by fixing the emission wavelength at 10 nm above its maximum and recording excitation from 350 nm to the maximum emission wavelength.
Photoactivation Rate of Photoactivatable Blue Fluorescent Protein.Under magnetic stirring, 2 mL of a pure and "dark" sample of PA-BFP 0.13 μM in PBS buffer was excited at 302 or 365 nm, recording the fluorescence at 445 nm during 1 h on the LS-55 fluorescence spectrometer from PerkinElmer, using an excitation slit of 15 nm and an emission slit of 5 nm.This instrument comes from the factory equipped with a high-energy pulsed xenon lamp.
Protein Analysis by Size Exclusion Chromatography.In total, 100 μL of each purified protein, at approximately 2 μg/μL, was independently analyzed on a Superdex 200 10/300 GL column from Pharmacia, using 0.1 M NaCl in 0.1 M phosphate buffer pH 7.2 as the mobile phase, at a flow rate of 0.75 mL/min.Detection was achieved by UV absorption at 280 nm.As shown in Figure S6, PA-BFP eluted as a monomer like its parental proteins sfGFP and SumireF.
Molecular Mass Determination.The molecular weight of PA-BFP in its dark and activated states was determined using ESI-MS-LC.The sample was applied in a system comprising an EASY-nLC II nanoflow pump (Thermo Fisher Co., San Jose, CA) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Co., San Jose, CA) with a nano-ESI source.The molecular mass of each sample was obtained by processing the data using the automatic deconvolution algorithm (Xtract RAW File).
Determination of the Quantum Yield and Molar Extinction Coefficient.The concentration of both activated proteins�PA-BFP and SumireF�was determined by the bicinchoninic acid assay using the QuantiPro Kit (Sigma− Aldrich), following the supplier's instructions, and bovine serum albumin as the standard.To determine the extinction coefficient, five dilutions of each protein were prepared in duplicate, and their absorbance was determined at their maximum absorbance wavelengths (Sumire: 342 nm, PA-BFP: 360 nm).Plotting concentration (μM) versus absorbance produces a straight line, the slope of which is the extinction coefficient in units μM −1 cm −1 .Each of the above dilutions was diluted 20 times with PBS buffer, and fluorescence was determined at the maximum emission wavelength (Sumire: 413 nm, PA-BFP: 445 nm), exciting each protein at its maximum absorption wavelength.As a reference for determining the quantum yield, we used pure enhanced GFP (EGFP), whose maximum excitation and emission wavelengths were centered at 490 and 517 nm, respectively.The reported quantum yield for EGFP is 0.60. 38ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c02603.Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico; orcid.org/0000-0001-9408-365X;Email: paul.gaytan@ibt.unam.mx

Figure 1 .
Figure 1.Phenotypical and sequence comparison of the photoactivatable blue fluorescent protein (PA-BFP) SumireF-Y66H.(A) Comparison of E. coli streaks expressing the protein PA-BFP and its precursor proteins SumireF and sfGFP, visualized at 302 nm over a UV transilluminator.(B) Sequence comparison of PA-BFP and its parental proteins Sumire, SumireF, and sfGFP.The chromophore-forming amino acids are shaded in gray, and the key amino acid change in PA-BFP is blue.

Figure 2 .
Figure 2. UV activation of the protein variant SumireF-Y66H (photoactivatable blue fluorescent protein [PA-BFP]).(A) Repetitive acquisition of fluorescence spectra for every 2 min of E. coli cells resuspended in PBS buffer expressing the initially dark protein.Excitation: 365 nm; scanning rate: 100 nm/min; excitation slit: 15 nm (maximum allowed); emission slit: 5 nm.(B) Photoactivation reaction of the pure dark protein PA-BFP in solution (0.13 μM) at two different excitation wavelengths, recording fluorescence at 445 nm.

Figure 3 .
Figure 3. Spectroscopic analysis of photoactivatable blue fluorescent protein (PA-BFP).(A) UV−vis absorbance of the pure protein PA-BFP before and after UV activation.(B) Excitation and emission spectra of the pure protein PA-BFP before and after UV activation.ExSp: excitation spectrum; EmSp: emission spectrum.

Figure 4 .
Figure 4. Proposed post-translational maturation mechanism of the chromophore found in the photoactivatable blue fluorescent protein (PA-BFP).The three chromophore-forming amino acids are Gly65, His66, and Gly67.Supporting results for each proposed structure are indicated on the right side.After UV activation of the dark intermediate, a water molecule and an additional proton were eliminated.Current research does not support which of the starting two steps, cyclization or oxidation, occurs first.