Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging
2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2023.102977
ISSN1083-351X
AutoresAndrey Nikolaev, Anna Yudenko, Anastasia Smolentseva, Andrey Bogorodskiy, Fedor Tsybrov, Valentin Borshchevskiy, Siarhei Bukhalovich, Vera V. Nazarenko, Elizaveta Kuznetsova, Oleg Semenov, Alina Remeeva, Ivan Gushchin,
Tópico(s)bioluminescence and chemiluminescence research
ResumoFlavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy. Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy. Flavins, such as riboflavin (RF, vitamin B2), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), are ubiquitous biomolecules present in virtually all living cells (1Edwards A.M. Structure and general properties of flavins.in: Weber S. Schleicher E. Flavins and Flavoproteins: Methods and Protocols. Springer, New York, NY2014: 3-13Crossref Scopus (41) Google Scholar, 2Pavlovska T. Cibulka R. Structure and properties of flavins.in: Flavin-Based Catalysis. John Wiley & Sons, Ltd, Weinheim, Germany2021: 1-27Crossref Scopus (5) Google Scholar). 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Most of the mutations described to date lead to minor hypsochromic shifts, with the exception of those in the singlet oxygen generator miniSOG2 protein (33Makhijani K. To T.-L. Ruiz-González R. Lafaye C. Royant A. Shu X. Precision optogenetic tool for selective single- and multiple-cell ablation in a live animal model system.Cell Chem. Biol. 2017; 24: 110-119Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Replacement of the conserved glutamine adjacent to FMN with a valine or a leucine results in a blue shift of ∼10 nm (34Wingen M. Potzkei J. Endres S. Casini G. Rupprecht C. Fahlke C. et al.The photophysics of LOV-based fluorescent proteins – new tools for cell biology.Photochem. Photobiol. Sci. 2014; 13: 875-883Crossref PubMed Google Scholar, 35Westberg M. Holmegaard L. Pimenta F.M. Etzerodt M. Ogilby P.R. Rational design of an efficient, genetically encodable, protein-encased singlet oxygen photosensitizer.J. Am. Chem. 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Nazarenko V.V. et al.The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins.J. Biol. Chem. 2021; 296: 100662Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Finally, mutation of flavin-adjacent asparagine can also lead to a 4 to 6 nm red shift (42Raffelberg S. Mansurova M. Gärtner W. Losi A. Modulation of the photocycle of a LOV domain photoreceptor by the hydrogen-bonding Network.J. Am. Chem. Soc. 2011; 133: 5346-5356Crossref PubMed Scopus (70) Google Scholar, 43Goncharov I.M. Smolentseva A. Semenov O. Natarov I. Nazarenko V.V. Yudenko A. et al.High-resolution structure of a naturally red-shifted LOV domain.Biochem. Biophys. Res. Commun. 2021; 567: 143-147Crossref PubMed Scopus (5) Google Scholar), and mutation of glutamine adjacent to the N1 atom can lead to a 4–8 nm red shift (44Kabir M.P. Ouedraogo D. Orozco-Gonzalez Y. Gadda G. Gozem S. Alternative strategy for spectral tuning of flavin-binding fluorescent proteins.J. Phys. Chem. B. 2023; 127: 1301-1311Crossref PubMed Scopus (2) Google Scholar). In this work, we set out to expand the range of color-tuned FbFPs and examine the possibility of fine tuning of flavin spectral properties. We tested the effects of random mutations of the conserved photoactive cysteine and two other amino acids on the properties of FbFP based on a thermostable LOV domain from Chloroflexus aggregans (CagFbFP). We obtained 22 variants with the emission maxima uniformly covering the spectral range from 486 nm (LOV domains with a glutamine to valine mutation) to 512 nm (observed for a double mutant I52V A85Q). To demonstrate the applicability of the resulting palette of FbFPs for multicolor imaging, we performed three-color imaging based on spectral separation and two-color fluorescence lifetime imaging with differentially labeled E. coli cultures, as well as two-color imaging of mammalian cells (HEK293T). FbFPs share a highly conservative fluorescence emission spectrum with two maxima at ∼500 and ∼525 nm, and a wide shoulder in the long wavelength region (Fig. 1). Previously, while searching for color-tuned variants, we analyzed the differences between the positions of absolute fluorescence emission maxima of mutated and parent proteins. However, we also identified variants with significantly deformed spectra, where the two emission maxima merge into one (see Fig. 2 for examples). For such variants, the nominal shift of the emission maximum does not correspond to the shift of the whole spectrum. Therefore, in addition to analyzing the position of the maximum, we also analyzed the perceived color of the emitted light (hue H in the HSV color representation), which reflects the overall position of the spectrum. For a more accurate determination of the position of maxima, we fitted the spectra with Gaussians (Fig. 1).Figure 2Examples of the fluorescence emission spectra of CagFbFP variants with different shapes. A, Two spectra that are overall shifted relative to each other but have similar positions of the emission maxima. B, Two spectra that are essentially not shifted relative to each other but have different positions of the nominal emission maxima. Positions of the emission maxima are denoted with dashed lines. CagFbFP, flavin-binding fluorescent protein based on LOV domain from Chloroflexus aggregans.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Most of natural LOV domains contain a photoactive cysteine (19Glantz S.T. Carpenter E.J. Melkonian M. Gardner K.H. Boyden E.S. Wong G.K.-S. et al.Functional and topological diversity of LOV domain photoreceptors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E1442-E1451Crossref PubMed Scopus (105) Google Scholar, 45Yee E.F. Diensthuber R.P. Vaidya A.T. Borbat P.P. Engelhard C. Freed J.H. et al.Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue.Nat. Commun. 2015; 6: 1-10Crossref Scopus (70) Google Scholar). In engineered LOVs, this cysteine has been replaced with alanine, glycine, serine, or proline. We reasoned that substituting it with some other amino acid may produce noticeable spectral alterations, given that the position is close to the flavin chromophore (Fig. 3A). Intriguingly, the effects of other substitutions have not been reported previously, and we decided to conduct site saturation mutagenesis for this position. To have a higher chance of obtaining a folded protein even after very destabilizing mutations, we chose the recently developed thermostable fluorescent reporter CagFbFP (46Nazarenko V.V. Remeeva A. Yudenko A. Kovalev K. Dubenko A. Goncharov I.M. et al.A thermostable flavin-based fluorescent protein from chloroflexus aggregans: a framework for ultra-high resolution structural studies.Photochem. Photobiol. Sci. 2019; 18: 1793-1805Crossref PubMed Google Scholar) as a template. We found that the majority of the colonies with random mutations A85→X either did not display altered spectra or lost fluorescent properties. Only emission of the variant A85Q was red-shifted for 10 nm, yet the protein had extremely low expression level and stability (melting temperature of 42 °C, ∼40 °C lower than that of CagFbFP). Given that the side chain of glutamine is bigger than that of native cysteine, we hypothesized that compensating mutations of the nearby I52 (Fig. 3A) to a polar or smaller residue might stabilize the variant A85Q. First, we produced double mutants A85Q I52T and A85Q I52V. Both mutations significantly increased the expression level and raised the thermal stability by ∼10 °C (Table 1). Site saturation mutagenesis I52→X performed on the A85Q variant produced mostly nonfluorescent cell colonies. Besides the mutations I52V and I52T, we identified the variant I52A A85Q, which has an untypical spectrum with the two emission peaks merging into one, along with low thermal stability (unfolding at 42 °C) and very low expression level.Table 1Main properties of the color-shifted CagFbFP variantsHueλexcaλexc is the position of the excitation maximum, λem is the position of the emission maximum, λfit1 and λfit2 are positions of the fitted Gaussians corresponding to the first and second peaks. (nm)λemaλexc is the position of the excitation maximum, λem is the position of the emission maximum, λfit1 and λfit2 are positions of the fitted Gaussians corresponding to the first and second peaks. (nm)λfit1aλexc is the position of the excitation maximum, λem is the position of the emission maximum, λfit1 and λfit2 are positions of the fitted Gaussians corresponding to the first and second peaks. (nm)λfit2aλexc is the position of the excitation maximum, λem is the position of the emission maximum, λfit1 and λfit2 are positions of the fitted Gaussians corresponding to the first and second peaks. (nm)Fluorescence lifetime τav, (ns)bThe errors are the standard deviations in three independent experiments. Tm1 and Tm2 are the temperatures of the first and second melting transitions, correspondingly.Tm1 (°C)bThe errors are the standard deviations in three independent experiments. Tm1 and Tm2 are the temperatures of the first and second melting transitions, correspondingly.Tm2 (°C)bThe errors are the standard deviations in three independent experiments. Tm1 and Tm2 are the temperatures of the first and second melting transitions, correspondingly.Expression (mg/l)Mutations154.14434864835133,76 ± 0,0571.4 ± 0.181.1 ± 0.3108Q148V153.64434874845143,90 ± 0,0573.4 ± 0.282.5 ± 0.3110Q148I152.74444884845153,93 ± 0,0471.7 ± 0.181.0 ± 0.2112Q148L151.04444914845123,24 ± 0,0361.1 ± 0.371.5 ± 0.2cThe third transition is observed at 47.0 °C ± 1.4 deg. C.21Q148H151.04454904855163,98 ± 0,1369.3 ± 0.579.0 ± 0.319Q148M149.24454944865153,88 ± 0,0960.6 ± 0.474.0 ± 0.514Q148S146.14484984885154,19 ± 0,0747.2 ± 0.859.0 ± 0.120Q148R144.44495044915174,51 ± 0,0855.9 ± 0.271.9 ± 0.310Q148E143.84484994915234,33 ± 0,1059.7 ± 0.473.2 ± 0.512Q148G142.64494974935244,16 ± 0,1057.5 ± 0.171.7 ± 0.38I52VA85S140.44504994945264,53 ± 0,0364.3 ± 0.278.1 ± 0.550Original protein137.24525004965294,68 ± 0,0656.7 ± 0.267.7 ± 0.215I52VA85V135.84505074955243,74 ± 0,1255.6 ± 0.28I52TA85QQ148C135.74525034965274,26 ± 0,0646.3 ± 0.258.9 ± 0.111I52TA85QQ148V135.04545034995263,66 ± 0,0755.8 ± 0.25I52VA85QQ148L134.64505114965213,67 ± 0,1755.0 ± 0.212I52TA85QQ148S134.64515064975263,86 ± 0,1052.4 ± 0.99I52TA85QQ148F134.04555024985314,40 ± 0,0549.6 ± 1.058.4 ± 0.834I52VA85QQ148V131.54535085005314,24 ± 0,0645.9 ± 0.559.0 ± 0.312I52TA85QQ148H130.54545065005334,30 ± 0,1147.4 ± 0.49I52VA85QQ148A129.34525125035364,57 ± 0,0748.6 ± 1.313I52VA85Q127.14555095025344,65 ± 0,0644.9 ± 0.156.7 ± 0.17I52TA85Q125.84545105025334,54 ± 0,0742.1 ± 0.18I52VA85QQ148Na λexc is the position of the excitation maximum, λem is the position of the emission maximum, λfit1 and λfit2 are positions of the fitted Gaussians corresponding to the first and second peaks.b The errors are the standard deviations in three independent experiments. Tm1 and Tm2 are the temperatures of the first and second melting transitions, correspondingly.c The third transition is observed at 47.0 °C ± 1.4 deg. C. Open table in a new tab We also tested the effects of mutations A85→X on the I52V variant of CagFbFP. No new variants with significant shifts were observed; I52V A85Q was obtained again, I52V A85S was slightly blue-shifted, and I52V A85V was slightly red-shifted compared to CagFbFP (Table 1). Previously, we tested the effects of substitutions of the conserved glutamine Q148 (Fig. 3A) with polar and charged amino acids (40Remeeva A. Nazarenko V.V. Kovalev K. Goncharov I.M. Yudenko A. Astashkin R. et al.Insights into the mechanisms of light-oxygen-voltage domain color tuning from a set of high-resolution X-ray structures.Proteins. 2021; https://doi.org/10.1002/prot.26078Crossref PubMed Scopus (7) Google Scholar), all of which displayed hypsochromic shifts reaching ∼6 nm. Here, we performed Q148→X random mutagenesis of CagFbFP and identified six new blue shifted variants with a maximum shift of 12 nm. Searching for more red-shifted variants, we also tested the effects of Q148→X mutagenesis on the already red-shifted variants I52V A85Q and I52T A85Q. Only one variant, I52T A85Q Q148N, gained an additional minor red shift, while nine other mutants uniformly covered the range of spectral shifts between it and the original CagFbFP. Based on the results of our site saturation mutagenesis rounds, we identified a palette of 22 variants, which, together with CagFbFP, uniformly cover the range of emission spectra with the maxima from 486 to 512 nm (Fig. 3, B and C and Table 1). The excitation spectra show less variation (Fig. 4). We have also measured fluorescence lifetime and thermal stability of the identified variants in vitro (Table 1). Previously, we reported proof-of-concept two-color fluorescence microscopy imaging with CagFbFP Q148K and CagFbFP I52T Q148K (41Röllen K. Granzin J. Remeeva A. Davari M.D. Gensch T. Nazarenko V.V. et al.The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins.J. Biol. Chem. 2021; 296: 100662Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). However, these two variants displayed low stability and low expression levels, making them unreliable for applications. Consequently, we decided to test whether the better variants, identified in this work, may be used for two- or three-color microscopy. We selected three mutants from the palette (Q148L, I52T A85Q, and the parent CagFbFP) based on their fluorescence emission maxima, expression levels, and thermal stability and expressed them independently in three E. coli cultures. The expression conditions were chosen so that the final fluorescence normalized to absorbance at 600 nm was within 20% for the three cultures, making it impossible to distinguish the cells based on fluorescence intensity. We recorded the fluorescence emission of monocultures as well as of the mixture of cells using twelve 9 nm channels in the range from 459 to 557 nm. Spectra from single variant cultures were used as a reference, and a linear unmixing procedure was used to determine the contribution of each of the reference spectra to the fluorescence emission of each pixel in the image of the mixture of cultures (Fig. 5). The cells harboring different variants could easily be distinguished from each other, which proves that three-color microscopy using FbFPs is possible. To demonstrate that the CagFbFP variants can also be distinguished when present in the same cell, as a proof of concept, we imaged HEK293T cells expressing CagFbFP in mitochondria and CagFbFP-Q148V in the cytoplasm. After linear unmixing, mitochondria can clearly be distinguished from the rest of the cell (Fig. 6). We also tested whether CagFbFP variants could be separated using fluorescence lifetime imaging microscopy (FLIM) in vivo. We expressed the variants Q148H and I52T A85Q, which differ both in emission maxima and in fluorescence lifetimes, in E. coli and obtained the microscopy images. The cultures could be distinguished based either on spectra or on fluorescence lifetime (Fig. 7), thus confirming the possibility of two-color FLIM using FbFPs. However, we note that the fluorescence lifetimes measured in vivo (∼2.95 ns and 3.75 ns) differed from those measured in vitro (3.24 ns and 4.65 ns) and displayed relatively high cell-to-cell variation; spectrum-based separation is less ambiguous than fluorescence lifetime-based separation. To further characterize the four CagFbFP variants employed for multicolor imaging, we determined their quantum yields and brightness of fluorescence (Table 2). We note that all of the variants have impaired fluorescence properties compared to the original protein, yet this does not preclude their applications (Figure 5, Figure 6, Figure 7).Table 2Optical properties of selected CagFbFP variantsMutationsExtinction coefficient, M−1 cm−1Quantum yieldBrightness, M−1 cm−1Original protein15,700aExtinction coefficient of the original protein measured using an alternative approach is 15,300 M−1 cm−1 (see Experimental procedures).0.345300Q148V15,7000.335200Q148L15,6000.304700Q148H15,1000.253800I52T A85Q14,3000.202900a Extinction coefficient of the original protein measured using an alternative approach is 15,300 M−1 cm−1 (see Experimental procedures). Open table in a new tab Previously, several attempts have been made to generate flavin-based fluorescent proteins with shifted spectra, with moderate success (33Makhijani K. To T.-L. Ruiz-González R. Lafaye C. Royant A. Shu X. 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In this work, we mutated a previously untargeted amino acid position, that of photoactive cysteine (corresponding to A85 in CagFbFP), in addition to mutations of other residues near the flavin (I52 and Q148, Fig. 3A), and identified a palette of 22 variants that uniformly cover the range of emission spectra with the maxima from 486 to 512 nm (Fig. 3C). Mutation A85Q produced a notable red shift but destabilized the protein and thus required compensatory mutations I52T or I52V. The effects of mutation A85Q appear to be roughly additive with the effects of substitutions of Q148. In particular, the mutation Q148V resulted in the largest hypsochromic shifts of the emission maxima for both CagFbFP and CagFbFP I52V A85Q. Interestingly, the variant I52V A85Q Q148V has a spectrum shape almost identical to unmutated CagFbFP, whereas that of I52V A85Q is significantly deformed. While previously only two-color imaging of bacteria using flavin-binding fluorescent proteins was reported (41Röllen K. Granzin J. Remeeva A. Davari M.D. Gensch T. Nazarenko V.V. et al.The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins.J. Biol. Chem. 2021; 296: 100662Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), here we demonstrate both two-color imaging of eukaryotic HEK293T cells (Fig. 6) and three-color imaging of bacteria (Fig. 5) employing the new variants. When expressed in E. coli, CagFbFP displays two melting transitions, corresponding to the protein molecules bound to FMN, and those bound to RF/FAD (20Smolentseva A. Goncharov I.M. Yudenko A. Bogorodskiy A. Semenov O. Nazarenko V.V. et al.Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species.Photochem. Photobiol. Sci. 2021; 20: 1645-1656Crossref PubMed Scopus (3) Google Scholar). Most mutants identified in this study also show two transitions. Variant Q148H, similarly to some other engineered variants (47Yudenko A. Smolentseva A. Maslov I. Semenov O. Goncharov I.M. Nazarenko V.V. et al.Rational design of a split flavin-based fluorescent reporter.ACS Synth. Biol. 2021; 10: 72-83Crossref PubMed Scopus (8) Google Scholar), displays three transitions. Some of the mutants are less stable and show only one transition that presumably corresponds to FMN-bound species, thus highlighting the utility of mutating an initially thermostable protein. Except for Q148H, all of the proteins refolded easily after thermal denaturation. We note that all of the red-shifted variants are significantly less stable compared to the original CagFbFP and have lower expression levels, whereas some of the blue-shifted variants (Q148V, Q148I, and Q148L) are stabilized and better expressed. Lifetime of fluorescence for the identified variants ranged from 3.24 ns for the Q148H variant to 4.68 ns for the I52V A85V variant, with no clear correlation with spectral properties (Table 1). The I52T A85Q variant has close to the highest lifetime of 4.65 ns, yet its emission spectrum is significantly different from that of Q148H, which has the lowest fluorescence lifetime. Thus, the pair Q148H and I52T A85Q could potentially be used for two-color imaging based on both spectral separation and fluorescence lifetime. Indeed, we show that two-color fluorescence lifetime-based imaging using CagFbFP variants is possible, although spectral unmixing results in better separation (Fig. 7). We note that fluorescence lifetime values as low as 3.17 ns and as high as 5.70 ns were previously reported for other FbFPs; probably, they can provide better separation (34Wingen M. Potzkei J. Endres S. Casini G. Rupprecht C. Fahlke C. et al.The photophysics of LOV-based fluorescent proteins – new tools for cell biology.Photochem. Photobiol. Sci. 2014; 13: 875-883Crossref PubMed Google Scholar). In this work, we showed that the range of fluorescence emission maxima positions of color-tuned flavoproteins can be expanded to 25 nm by mutating the previously untested position, that of the photoactive cysteine of FbFP. The position of the emission maximum can also be finely tuned by mutating the conserved glutamine. The developed palette of fluorescent proteins, differing by one, two, or three amino acids, can be used for further understanding of the mechanisms of flavin color tuning (48Mroginski M.-A. Adam S. Amoyal G.S. Barnoy A. Bondar A.-N. Borin V.A. et al.Frontiers in multiscale modeling of photoreceptor proteins.Photochem. Photobiol. 2021; 97: 243-269Crossref PubMed Scopus (18) Google Scholar). The obtained variants can be used for three-color imaging based on spectral separation and two-color fluorescence lifetime imaging, paving the way for further applications of FbFPs in fluorescence microscopy.
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