A Unique Genetically Encoded FRET Pair in Mammalian Cells

Förster resonance energy transfer (FRET) between two suitable fluorophores is a powerful tool to monitor dynamic changes in protein structure in vitro and in vivo. The ability to genetically encode a FRET pair represents a convenient “labeling‐free” strategy to incorporate them into target protein(s). Currently, the only genetically encoded FRET pairs available for use in mammalian cells use fluorescent proteins. However, their large size can lead to unfavorable perturbations, particularly when two are used at the same time. Additionally, fluorescent proteins are largely restricted to a terminal attachment to the target, which might not be optimal. Here, we report the development of an alternative genetically encoded FRET pair in mammalian cells that circumvents these challenges by taking advantage of a small genetically encoded fluorescent unnatural amino acid as the donor and enhanced green fluorescent protein (EGFP) as the acceptor. The small size of Anap relative to fluorescent proteins, and the ability to co‐translationally incorporate it into internal sites on the target protein, endows this novel FRET pair with improved versatility over its counterparts that rely upon two fluorescent proteins.     

Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro

In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target—green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker—were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.     

Self-reporting materials: protein-mediated visual indication of damage in a bulk polymer

Damage self-reporting materials are able to indicate the presence of microscopic damaged regions by easy to detect signals, such as fluorescence. Therefore, these smart materials can reduce the risk of catastrophic failure of load-bearing components, e.g., in aerospace and construction applications. We highlight here our proof-of-concept paper and we present some additional data, which shows that proteins can be used as mechanophores in solid polymeric materials. Macroscopic mechanical forces were transferred from the polymer to the embedded proteins. The biomolecules act as molecular strain sensor, giving the material the desired self-reporting property. Poly(ethylene glycol) and poly(acrylamide) (PAAm) networks were doped with small amounts of thermsosome (THS), a protein cage from the family of chaperonins, that encapsulated a pair of fluorescent proteins. THS acts as a scaffold which brings the two fluorescent proteins into distance suitable for fluorescence resonance energy transfer (FRET). Moreover, THS can be distorted by mechanic forces so that the distance between the fluorescent proteins changes, leading to a change in FRET efficiency. Using the brittle PAAm as a model system, we were able to visualize microcracks in the polymers by FRET microscopy and by fluorescence lifetime imaging. THS also stabilizes the encapsulated guest proteins against thermal denaturation, increasing their half-live at 70 degrees C by a factor of 2.3.     

Optimized fluorescent trimethoprim derivatives for in vivo protein labeling

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.     

A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome**

Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.     

Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.     

Photoinducible bioorthogonal chemistry: a spatiotemporally controllable tool to visualize and perturb proteins in live cells

Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from. In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole-alkene cycloaddition reaction ("photoclick chemistry") and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M(-1) s(-1)), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short "bridge" between two flanking phenyl rings. This photoclick chemistry has been used to label proteins rapidly (within ～1 min) both in vitro and in E. coli . To create an effective interface with biology, we have identified both a metabolically incorporable alkene amino acid, homoallylglycine, and a genetically encodable tetrazole amino acid, p-(2-tetrazole)phenylalanine. We demonstrate the utility of these two moieties, respectively, in spatiotemporally controlled imaging of newly synthesized proteins and in site-specific labeling of proteins. Additionally, we demonstrate the use of the photoclick chemistry to perturb the localization of a fluorescent protein in mammalian cells.     

Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells

Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.     

Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation

A screen of Trp37 mutants of Escherichia coli lipoic acid ligase (LplA) revealed enzymes capable of ligating an aryl-aldehyde or aryl-hydrazine substrate to LplA's 13-residue acceptor peptide. Once site-specifically attached to recombinant proteins fused to this peptide, aryl-aldehydes could be chemoselectively derivatized with hydrazine-probe conjugates, and aryl-hydrazines could be derivatized in an analogous manner with aldehyde-probe conjugates. Such two-step labeling was demonstrated for AlexaFluor568 targeting to monovalent streptavidin in vitro, and to neurexin-1β on the surface of living mammalian cells. To further highlight this technique, we labeled the low-density lipoprotein receptor on the surface of live cells with fluorescent phycoerythrin protein to allow single-molecule imaging and tracking over time.     

Designed Proteins as Novel Imaging Reagents in Living Escherichia coli

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co‐expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs.     

The mRubyFT Protein,Genetically Encoded Blue-to-Red Fluorescent Timer

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2–3- and 5–7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.     

Recent Progress in Strategies for the Creation of Protein‐Based Fluorescent Biosensors

The creation of novel bioanalytical tools for the detection and monitoring of a range of important target substances and biological events in vivo and in vitro is a great challenge in chemical biology and biotechnology. Protein‐based fluorescent biosensors—integrated devices that convert a molecular‐recognition event to a fluorescent signal—have recently emerged as a powerful tool. As the recognition units various proteins that can specifically recognize and bind a variety of molecules of biological significance with high affinity are employed. For the transducer, fluorescent proteins, such as green fluorescent protein (GFP) or synthetic fluorophores, are mostly adopted. Recent progress in protein engineering and organic synthesis allows us to manipulate proteins genetically and/or chemically, and a library of such protein scaffolds has been significantly expanded by genome projects. In this review, we briefly describe the recent progress of protein‐based fluorescent biosensors on the basis of their platform and construction strategy, which are primarily divided into the genetically encoded fluorescent biosensors and chemically constructed biosensors.     

His-tags as Zn(II) binding motifs in a protein-based fluorescent sensor

Fluorescent indicators that allow real-time imaging of Zn(II) in living cells are invaluable tools for understanding Zn(II) homeostasis. Genetically encoded sensors based on fluorescence resonance energy transfer between fluorescent protein domains have important advantages over synthetic probes. We discovered that hexahistidine tags have a strong tendency to dimerize upon binding of Zn(II) in solution and we used this principle to develop a new protein-based sensor for Zn(II). Enhanced cyan and yellow fluorescent proteins were connected by long flexible peptide linkers and His-tags were incorporated at both termini of this fusion protein. The resulting sensor CLY9-2His allows the ratiometric fluorescent detection of Zn(II) in the nanomolar range. In addition, CLY9-2His is selective over the physiologically relevant metal ions Fe(II), Mn(II), Ca(II) and Mg(II). Our approach demonstrates the potential of using small peptides as metal-binding ligands in chelating fluorescent protein chimeras.     

Exploration of biarsenical chemistry--challenges in protein research

The fluorescent modification of proteins (with genetically encoded low-molecular-mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent-labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6- to 12-residue), appropriately spaced, tetracysteine sequence (-CCXXCC-); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact-cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein-protein interaction studies, protein stability, and aggregation studies.     

Protein Microarray Copying: Easy on-Demand Protein Microarray Generation Compatible with Fluorescence and Label-Free Real-Time Analysis

Protein microarrays are essential to understand complex protein interaction networks. Their production, however, is a challenge and renders this technology unattractive for many laboratories. Recent developments in cell-free protein microarray generation offer new opportunities, but are still expensive and cumbersome in practice. Herein, we describe a cost-effective and user-friendly method for the cell-free production of protein microarrays. From a polydimethylsiloxane (PDMS) flow cell containing an expressible DNA microarray, proteins of interest are synthesised by cell-free expression and then immobilised on a capture surface. The resulting protein microarray can be regarded as a “copy” of the DNA microarray. 2 His₆- and Halo-tagged fluorescent reference proteins were used to demonstrate the functionality of nickel nitrilotriacetic acid (Ni-NTA) and Halo-bind surfaces in this copy system. The described process can be repeated several times on the same DNA microarray. The identity and functionality of the proteins were proven during the copy process by their fluorescence and on the surface through a fluorescent immune assay. Also, single-colour reflectometry (SCORE) was applied to show that, on such copied arrays, real-time binding kinetic measurements were possible.     

Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One‐ and Two‐Photon Illumination

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen‐OH, a photochemically stable inducer of the receptor specific for 4‐hydroxy‐tamoxifen (ER^T2). Cyclofen‐OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER^T2 receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen‐OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two‐photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.     

Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging

A reengineered human cellular retinol binding protein II (hCRBPII), a 15-kDa protein belonging to the intracellular lipid binding protein (iLBP) family, generates a highly fluorescent red pigment through the covalent linkage of a merocyanine aldehyde to an active site lysine residue. The complex exhibits “turn-on” fluorescence, due to a weakly fluorescent aldehyde that “lights up” with subsequent formation of a strongly fluorescent merocyanine dye within the binding pocket of the protein. Cellular penetration of merocyanine is rapid, and fluorophore maturation is nearly instantaneous. The hCRBPII/merocyanine complex displays high quantum yield, low cytotoxicity, specificity in labeling organelles, and compatibility in both cancer cell lines and yeast cells. The hCRBPII/merocyanine tag is brighter than most common red fluorescent proteins.     

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging α(v)β(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.     

LSSmScarlet,dCyRFP2s,dCyOFP2s and CRISPRed2s,Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.     

A Luminescent Cyclometalated Iridium(III) Complex Accumulates in Mitochondria and Induces Mitochondrial Shortening by Conjugation to Specific Protein Targets

We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)₂(phen‐ITC)](PF₆) (Ir‐ITC; Hpq=2‐phenylquinoline, phen‐ITC=5‐isothiocyanate‐1,10‐phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir‐ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir‐ITC with live‐cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two‐dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage‐dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir‐ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.