Green Fluorescent Protein Variants Research Articles

Genetically Encoded Biosensors for the Fluorescence Detection of O2 and Reactive O2 Species.

The intracellular concentrations of oxygen and reactive oxygen species (ROS) in living cells represent critical information for investigating physiological and pathological conditions. Real-time measurement often relies on genetically encoded proteins that are responsive to fluctuations in either oxygen or ROS concentrations. The direct binding or chemical reactions that occur in their presence either directly alter the fluorescence properties of the binding protein or alter the fluorescence properties of fusion partners, mostly consisting of variants of the green fluorescent protein. Oxygen sensing takes advantage of several mechanisms, including (i) the oxygen-dependent hydroxylation of a domain of the hypoxia-inducible factor-1, which, in turn, promotes its cellular degradation along with fluorescent fusion partners; (ii) the naturally oxygen-dependent maturation of the fluorophore of green fluorescent protein variants; and (iii) direct oxygen binding by proteins, including heme proteins, expressed in fusion with fluorescent partners, resulting in changes in fluorescence due to conformational alterations or fluorescence resonance energy transfer. ROS encompass a group of highly reactive chemicals that can interconvert through various chemical reactions within biological systems, posing challenges for their selective detection through genetically encoded sensors. However, their general reactivity, and particularly that of the relatively stable oxygen peroxide, can be exploited for ROS sensing through different mechanisms, including (i) the ROS-induced formation of disulfide bonds in engineered fluorescent proteins or fusion partners of fluorescent proteins, ultimately leading to fluorescence changes; and (ii) conformational changes of naturally occurring ROS-sensing domains, affecting the fluorescence properties of fusion partners. In this review, we will offer an overview of these genetically encoded biosensors.

Protein stability governed by α1-2 helices in Pvr4 is essential for localization and cell death.

Plant nucleotide-binding domain leucine-rich-repeat receptor (NLR) confers disease resistance to various pathogens by recognizing effectors derived from the pathogen. Previous studies have shown that overexpression of the CC domain in several NLRs triggers cell death, implying that the CC domain plays an important role as a signaling module. However, how CC domain transduces immune signals remains largely unknown. A Potyvirus-resistant NLR protein, Pvr4, possesses a CC domain (CCPvr4 ) that induces cell death upon transient overexpression in Nicotiana benthamiana. In this study, loss-of-function mutants were generated by error-prone PCR-based random mutagenesis to understand the molecular mechanisms underlying CCPvr4 -mediated cell death. Cell biology and biochemical studies revealed that M16 and Q52 in the α1 and α2 helices, respectively, are crucial for protein stability, and mutation of these residues disrupts localization to the plasma membrane and oligomerization activity. The increase of the protein stability of these mutants by tagging a green fluorescent protein (GFP) variant led to restoration of cell death-inducing activity and plasma membrane localization. Another mutant, I7E in the very N-terminal region, lost cell death-inducing activity by weakening the interaction with plasma membrane H+ -ATPase compared to CCPvr4 , although the protein remained in the plasma membrane. Moreover, most of the mutated residues are on the outer surface of the funnel shape in the predicted pentameric CCPvr4 , implying that the disordered N-terminal region plays a crucial role in association with PMA as well as targeting to the plasma membrane. This work could provide insights into the molecular mechanisms of cell death induced by NLR immune receptors.

Tuning Electrostatic Interactions To Control Orientation of GFP Protein Adsorption on Silica Surface.

The adsorption of green fluorescent protein (GFP) on silica surfaces has been the subject of growing interest due to its potential applications in various fields, including biotechnology and biomedicine. In this study, we used all-atom molecular dynamics simulations to investigate the charge-driven adsorption of wild type GFP and its supercharged variants on silica surfaces. The results showed that the positively charged variant of GFP adsorbed on the negatively charged silica surface with minimal loss in its secondary structure. Further studies were conducted to understand the role of surface charge distribution on two other positively charged variants of GFP, and the results showed that the orientation of GFP on silica can be easily tuned by careful mutations of the charged amino acid residues on the GFP. This study provides valuable molecular insights into the role of electrostatic-driven adsorption of GFP and highlights the importance of charge interactions in the adsorption process.

Fusion proteins of organophosphorus hydrolase and pHluorin for a whole-cell biosensor for organophosphorus pesticide measurement.

Fusion proteins composed of an organophosphorus hydrolase (OPH) and pHluorin, a pH-sensitive green fluorescent protein variant, were constructed as whole-cell biosensors to measure organophosphorus pesticides. pHluorin was used to detect the pH changes because of the hydrolase of paraoxon by OPH. To examine the order of fusion of OPH and pHluorin, pHluorin-OPH and OPH-pHluorin fusion proteins were constructed. In addition, a peptide linker consisting of 15 amino acid was inserted between pHluorin and OPH to reduce steric hindrance. OPH and pHluorin activities were evaluated in cells expressing the four fusion proteins. The both activities of pHluorin-OPH and pHluorin-linker-OPH were higher than that of OPH-pHluorin and OPH-linker-pHluorin. Effects of the peptide linker on the activities were slight. Therefore, pHluorin-OPH and pHluorin-linker-OPH were found to be suitable for organophosphorus pesticide measurements. Using cells expressing pHluorin-linker-OPH, 0.5μg/mL of paraoxon could be measured.

Use of the redox-dependent intein system for enhancing production of the cyclic green fluorescent protein

To expand the reported redox-dependent intein system application, in this work, we used the split intein variant with highly trans-splicing efficiency and minimal extein dependence to cyclize the green fluorescent protein variant reporter in vitro. The CPG residues were introduced adjacent to the intein's catalytic cysteine for reversible formation of a disulfide bond to retard the trans-splicing reaction under the oxidative environment. The cyclized reporter protein in Escherichia coli cells was easily prepared by organic extraction and identified by the exopeptidase digestion. The amounts of extracted cyclized protein reporter in BL21 (DE3) cells were higher than those in hyperoxic SHuffle T7 coexpression system for facilitating the disulfide bond formation. The double His6-tagged precursor was purified for in vitro cyclization of the protein for 3 h. Compared with the purified linear counterpart, the cyclic reporter showed about twofold increase in fluorescence intensity, exhibited thermal and hydrolytic stability, and displayed better folding efficiency in BL21 (DE3) cells at the elevated temperature. Taken together, the developed redox-dependent intein system will be used for producing other cyclic disulfide-free proteins. The cyclic reporter is a potential candidate applied in certain thermophilic aerobes.

Global Analysis of Multi-Mutants to Improve Protein Function

The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants. We have applied GMMA to a previously published experiment reporting on >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1–15 amino acid substitutions (Sarkisyan et al., 2016). The GMMA method achieves a good fit to this dataset while being analytically transparent. We show experimentally that the six top-ranking substitutions progressively enhance GFP. More broadly, using only a single experiment as input our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function. In conclusion, we suggest that large libraries of multiply-substituted variants may provide a unique source of information for protein engineering.

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors.

Selenium is a component of selenoproteins, which plays a crucial role in cellular redox homeostasis, thyroid metabolism, and DNA synthesis. Selenium has pleiotropic effects like antioxidant and anti-inflammatory activities; however, excess intake of selenium can imbalance such processes. The effects of selenium on human health are numerous and complex, demanding additional research to monitor the flux rate of selenium. Here, we have created a noninvasive and highly efficient genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor, SelFS (Selenium FRET-Sensor), for real-time monitoring of selenium at the cellular and subcellular levels. The construct of the nanosensor contains a selenium-binding protein (SeBP) as the selenium-detecting element inserted between the green fluorescent protein variants enhanced cyan fluorescent protein and Venus. In the presence of selenium, SelFS brings a conformational change, which is seen in the form of FRET. In vitro studies showed that SelFS is highly specific and selective for selenium and stable at an altered pH range from 5.0 to 8.0. SelFS is a flexible and dynamic tool for the detection of selenium in both prokaryotes and eukaryotes in a noninvasive way, with a binding constant (K d) of 0.198 × 10-6 M as compared to its mutants. The developed nanosensor can provide us a reporter tool for a wide range of industrial and environmental applications, which will help us to understand its functions in biological systems.

Disulfide Bond-mediated Oligomerization of a Green Fluorescent Protein in Solution

A green fluorescent protein (GFP) variant with two cysteine residues added to the protein surface was modified with 2,2′-dipyridyl disulfide and oligomerization of the modified protein and the unmodified variant via the disulfide bond was achieved in solution. The fluorescence anisotropy decay of the obtained GFP oligomer is capable of efficient homo-FRET.

Structural origin and rational development of bright red noncanonical variants of green fluorescent protein.

The incorporation of noncanonical amino acids (ncAAs) into fluorescent proteins is promising for red-shifting their fluorescence and benefiting tissue imaging with deep penetration and low phototoxicity. However, ncAA-based red fluorescent proteins (RFPs) have been rare. The 3-aminotyrosine modified superfolder green fluorescent protein (aY-sfGFP) represents a recent advance, yet the molecular mechanism for its red-shifted fluorescence remains elusive while its dim fluorescence hinders applications. Herein, we implement femtosecond stimulated Raman spectroscopy to obtain structural fingerprints in the electronic ground state and reveal that aY-sfGFP possesses a GFP-like instead of RFP-like chromophore. Red color of aY-sfGFP intrinsically arises from a unique "double-donor" chromophore structure that raises ground-state energy and enhances charge transfer, notably differing from the conventional conjugation mechanism. We further developed two aY-sfGFP mutants (E222H and T203H) with significantly improved (∼12-fold higher) brightness by rationally restraining the chromophore's nonradiative decay through electronic and steric effects, aided by solvatochromic and fluorogenic studies of the model chromophore in solution. This study thus provides functional mechanisms and generalizable insights into ncAA-RFPs with an efficient route for engineering redder and brighter fluorescent proteins.

Physiological jump in erythrocyte redox potential during Plasmodium falciparum development occurs independent of the sickle cell trait

The redox state of the host-parasite unit has been hypothesized to play a central role for the fitness of the intraerythrocytic blood stages of the human malaria parasite Plasmodium falciparum. In particular, hemoglobinopathies have been suggested to cause a more oxidizing environment, thereby protecting from severe malaria. Here we determined the redox potential of infected wild-type (hemoglobin AA) or sickle trait (hemoglobin AS) erythrocytes using parasite-encoded variants of the redox-sensitive green-fluorescent protein 2 (roGFP2). Our non-invasive roGFP2 single-cell measurements revealed a reducing steady-state redox potential of −304 ± 11 mV for the erythrocyte cytosol during ring-stage development and a rather sudden oxidation to −278 ± 12 mV during trophozoite-stage development around 28 h post invasion. There was no significant difference between wild-type or sickle trait erythrocytes regarding the stage dependence and the detected increase of the redox potential during the intraerythrocytic life cycle. The steady-state redox potential of the parasite cytosol, between −304 and −313 mV, was highly reducing throughout the life cycle. The redox potential in the parasitophorous vacuole at the interface between the secretory pathway and the erythrocyte was −284 ± 10 mV and remained stable during trophozoite-stage development with implications for the export of disulfide-containing proteins. In summary, P. falciparum blood stage development from the late ring to the early trophozoite stage causes a physiological jump in erythrocyte redox potential irrespective of the presence or absence of hemoglobin S.

Understanding Supramolecular Assembly of Supercharged Proteins.

Ordered supramolecular assemblies have recently been created using electrostatic interactions between oppositely charged proteins. Despite recent progress, the fundamental mechanisms governing the assembly of oppositely supercharged proteins are not fully understood. Here, we use a combination of experiments and computational modeling to systematically study the supramolecular assembly process for a series of oppositely supercharged green fluorescent protein variants. We show that net charge is a sufficient molecular descriptor to predict the interaction fate of oppositely charged proteins under a given set of solution conditions (e.g., ionic strength), but the assembled supramolecular structures critically depend on surface charge distributions. Interestingly, our results show that a large excess of charge is necessary to nucleate assembly and that charged residues not directly involved in interprotein interactions contribute to a substantial fraction (∼30%) of the interaction energy between oppositely charged proteins via long-range electrostatic interactions. Dynamic subunit exchange experiments further show that relatively small, 16-subunit assemblies of oppositely charged proteins have kinetic lifetimes on the order of ∼10–40 min, which is governed by protein composition and solution conditions. Broadly, our results inform how protein supercharging can be used to create different ordered supramolecular assemblies from a single parent protein building block.

\(\textit{n}\)-alkanol stress-induced cell envelope injury of \(σ^{E}\) promoter in \(\textit{Escherichia coli}\)

To characterize the cellular stress by n-alkanols with different alkyl chain lengths in Escherichia coli, we investigated how n-alkanols damage cell envelope permeability and whether they enhance the promoter activity of the envelope stress response regulator, σE, by using variants of green fluorescent protein (GFP). By using E. coli cells having GFPuv expressing and localizing in the cytoplasm, the inner membrane, and the periplasm, after exposure to n-alkanols, the fluorescent intensity of GFPuv released from cells was examined. Our data showed that at the similar levels of cell death of about 90–97%, ethanol, a short-chain alkanol, at a concentration of 20% damaged the outer membrane more greatly than the inner membrane, whereas a longer-chain alkanol of pentanol at a concentration of 1.125% damaged both of the outer and inner membranes. Then we investigated the envelope stress response to n-alkanols by σE factor by ratiometric analysis of rpoE promoter activity for the downstream GFPuv expression referenced to that of housekeeping sigma 70 (σ70 ) recognizing lacUV5 promoter for red fluorescent protein (RFP) expression. The results indicated that the relative activity of rpoE promoter by pentanol was much greater than that of ethanol. The degree of its sensitization by rpoE deficiency was much more remarkable for cells treated with pentanol than for those with ethanol. The results suggest that the response of the σE plays a significant role in the membrane integrity and survival of E. coli cells treated with n-alkanols depending on the alkyl chain length of the molecule.

Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant.

Aims:Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2).Results:SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) in cellulo. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI in cellulo.Innovation:This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited.Conclusion:Improved structural stability of sfroGFP2 gives rise to increased FI in cellulo. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. Antioxid. Redox Signal. 37, 1–18.

GREEN FLUORESCENT PROTEIN variants with enhanced folding are more efficiently imported into chloroplasts.

Chloroplasts and mitochondria are subcellular organelles that evolved from cyanobacteria and α-proteobacteria, respectively. Although they have their own genomes, the majority of their proteins are encoded by nuclear genes, translated by cytosolic ribosomes, and imported via outer and inner membrane translocon complexes. The unfolding of mature regions of proteins is thought to be a prerequisite for the import of the proteins into these organelles. However, it is not fully understood how protein folding properties affect their import into these organelles. In this study, we examined the import behavior of chloroplast and mitochondrial reporters with normal green fluorescent protein (GFP) and two GFP variants with enhanced folding propensity, superfolder GFP (sfGFP) and extra-superfolder GFP (esGFP), which is folded better than sfGFP. sfGFP and esGFP were less dependent on the sequence motifs of the transit peptide (TP) and import machinery during protein import into Arabidopsis (Arabidopsis thaliana) chloroplasts, compared with normal GFP. sfGFP and esGFP were efficiently imported into chloroplasts by a mutant TP with an alanine substitution in the N-terminal MLM motif, whereas the same mutant TP showed a defect in importing normal GFP into chloroplasts. Moreover, sfGFP and esGFP were efficiently imported into plastid protein import 2 (ppi2) and heat shock protein 93-V (hsp93-V) plants, which have mutations in atToc159 and Hsp93-V, respectively. In contrast, the presequence-mediated mitochondrial import of sfGFP and esGFP was severely impaired. Based on these results, we propose that the chloroplast import machinery is more tolerant to different folding states of preproteins, whereas the mitochondrial machinery is more specialized in the translocation of unfolded preproteins.

Mix and Match: Promoters and Terminators for Tuning Gene Expression in the Methylotrophic Yeast Ogataea polymorpha.

The yeast Ogataea polymorpha is an upcoming host for bio-manufacturing due to its unique physiological properties, including its broad substrate spectrum, and particularly its ability to utilize methanol as the sole carbon and energy source. However, metabolic engineering tools for O. polymorpha are still rare. In this study we characterized the influence of 6 promoters and 15 terminators on gene expression throughout batch cultivations with glucose, glycerol, and methanol as carbon sources as well as mixes of these carbon sources. For this characterization, a short half-life Green Fluorescent Protein (GFP) variant was chosen, which allows a precise temporal resolution of gene expression. Our promoter studies revealed how different promoters do not only influence the expression strength but also the timepoint of maximal expression. For example, the expression strength of the catalase promoter (pCAT) and the methanol oxidase promoter (pMOX) are comparable on methanol, but the maximum expression level of the pCAT is reached more than 24 h earlier. By varying the terminators, a 6-fold difference in gene expression was achieved with the MOX terminator boosting gene expression on all carbon sources by around 50% compared to the second-strongest terminator. It was shown that this exceptional increase in gene expression is achieved by the MOX terminator stabilizing the mRNA, which results in an increased transcript level in the cells. We further found that different pairing of promoters and terminators or the expression of a different gene (β-galactosidase gene) did not influence the performance of the genetic parts. Consequently, it is possible to mix and match promoters and terminators as independent elements to tune gene expression in O. polymorpha.

Pull-Down Into Active Inclusion Bodies and Their Application in the Detection of (Poly)-Phosphates and Metal-Ions.

Inclusion bodies are typically ignored as they are considered unwanted protein waste generated by prokaryotic host cells during recombinant protein production or harmful protein inclusions in human cell biology. However, these protein particles may have applications for in vivo immobilization in industrial biocatalysis or as cell-tolerable protein materials for the pharmaceuticals industry and clinical development. Thus, there is a need to in vivo “pull-down” (insolubilize) soluble enzymes and proteins into inclusion bodies. Accordingly, in this study, sequences from the short-chain polyphosphatase ygiF were used to design pull-down tags capable of detecting (poly)-phosphates and metal ions. These tags were compared with the entire CHAD domain from Escherichia coli ygiF and SACS2 CHAD from Saccharolobus solfataricus. The results demonstrated that highly soluble green fluorescent protein variants could be pulled down into the inclusion bodies and could have modified sensitivity to metals and di-/tri-inorganic phosphates.

Engineering a Fluorescent Protein Color Switch Using Entropy-Driven β-Strand Exchange.

Protein conformational switches are widely used in biosensing. They are often composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals via conformational changes. Here, we create a biosensor in which binding-induced folding of the input domain drives a conformational shift in the output domain that results in a sixfold green-to-yellow ratiometric fluorescence change in vitro and a 35-fold intensiometric fluorescence increase in cultured cells. The input domain consists of circularly permuted FK506 binding protein (cpFKBP) that folds upon binding its target ligand (FK506 or rapamycin). cpFKBP folding induces the output domain, an engineered green fluorescent protein (GFP) variant, to replace one of its β-strands (containing T203 and specifying green fluorescence) with a duplicate β-strand (containing Y203 and specifying yellow fluorescence) in an intramolecular exchange reaction. This mechanism employs the loop-closure entropy principle, embodied by the folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This study highlights the high-energy barriers present in GFP folding which cause β-strand exchange to be slow and are also likely responsible for the shift from the β-strand exchange mechanism in vitro to ligand-induced chromophore maturation in cells. The proof-of-concept design has the advantages of full genetic encodability and potential for modularity. The latter attribute is enabled by the natural coupling of binding and folding and circular permutation of the input domain, which theoretically allows different binding domains to be compatible for insertion into the GFP surface loop.

Predicting mutant outcome by combining deep mutational scanning and machine learning.

Deep mutational scanning provides unprecedented wealth of quantitative data regarding the functional outcome of mutations in proteins. A single experiment may measure properties (eg, structural stability) of numerous protein variants. Leveraging the experimental data to gain insights about unexplored regions of the mutational landscape is a major computational challenge. Such insights may facilitate further experimental work and accelerate the development of novel protein variants with beneficial therapeutic or industrially relevant properties. Here we present a novel, machine learning approach for the prediction of functional mutation outcome in the context of deep mutational screens. Using sequence (one-hot) features of variants with known properties, as well as structural features derived from models thereof, we train predictive statistical models to estimate the unknown properties of other variants. The utility of the new computational scheme is demonstrated using five sets of mutational scanning data, denoted "targets": (a) protease specificity of APPI (amyloid precursor protein inhibitor) variants; (b-d) three stability related properties of IGBPG (immunoglobulin G-binding β1 domain of streptococcal protein G) variants; and (e) fluorescence of GFP (green fluorescent protein) variants. Performance is measured by the overall correlation of the predicted and observed properties, and enrichment-the ability to predict the most potent variants and presumably guide further experiments. Despite the diversity of the targets the statistical models can generalize variant examples thereof and predict the properties of test variants with both single and multiple mutations.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein.

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

Site-Specific Fragmentation of Green Fluorescent Protein Induced by Blue Light.

Green fluorescent protein (GFP) and related fluorescent proteins have multiple applications in cell biology, and elucidating their functions has been at the focus of biophysical research for about three decades. Fluorescent proteins can be bleached by intense irradiation, and a number of them undergo photoconversion. Rare cases have been reported where distant functional relatives of GFP exhibit UV-light-induced protein fragmentation. Here, we show that irreversible bleaching of two different variants of GFP (sfGFP, EGFP) with visible light is paralleled by successive backbone fragmentation of the protein. Mass spectrometry revealed that the site of fragmentation resides at the fluorophore, between residue positions 65 and 66.