Fluorogen-activating Protein Research Articles

Leaflet specific phospholipid imaging using genetically encoded proximity sensors.

The lipid composition of cells varies widely across organelles and between individual membrane leaflets. Transport proteins acting within and between membranes are thought to generate this heterogeneity, but measuring their functions has been hampered by limited tools for imaging lipid composition at relevant spatial resolutions. Here we present fluorogen-activating coincidence sensing (FACES), a chemogenetic tool capable of quantitatively imaging subcellular lipid pools and reporting their transbilayer orientation in living cells. FACES combines bioorthogonal chemistry with genetically encoded fluorogen-activating proteins (FAPs) for reversible proximity sensing of conjugated molecules. We first apply this approach to identify roles for lipid transfer proteins (LTP) that traffic phosphatidylcholine pools between the ER and mitochondria. We then show that transmembrane domain-containing FAPs can be used to reveal the membrane asymmetry of multiple lipid classes that is generated at the trans-Golgi network (TGN). Using FACES, we show how the asymmetry of a phosphatidylserine analogue at the TGN is supported by the activity of both cytosolic LTPs and transmembrane flippases. Lastly, we demonstrate that FACES is a generalizable tool for subcellular detection of other molecule classes by measuring changes in mitochondrial N -acetylhexosamine levels. These results introduce the application of fluorogenic tags for spatially-defined molecular imaging.

Biofilm dispersal patterns revealed using far-red fluorogenic probes.

Bacteria frequently colonize niches by forming multicellular communities called biofilms. To explore new territories, cells exit biofilms through an active process called dispersal. Biofilm dispersal is essential for bacteria to spread between infection sites, yet how the process is executed at the single-cell level remains mysterious due to the limitations of traditional fluorescent proteins, which lose functionality in large, oxygen-deprived biofilms. To overcome this challenge, we developed a cell-labeling strategy utilizing fluorogen-activating proteins (FAPs) and cognate far-red dyes, which remain functional throughout biofilm development, enabling long-term imaging. Using this approach, we characterize dispersal at unprecedented resolution for the global pathogen Vibrio cholerae. We reveal that dispersal initiates at the biofilm periphery and approximately 25% of cells never disperse. We define novel micro-scale patterns that occur during dispersal, including biofilm compression during cell departure and regional heterogeneity in cell motions. These patterns are attenuated in mutants that reduce overall dispersal or that increase dispersal at the cost of homogenizing local mechanical properties. Collectively, our findings provide fundamental insights into the mechanisms of biofilm dispersal, advancing our understanding of how pathogens disseminate. Moreover, we demonstrate the broad applicability of FAPs as a powerful tool for high-resolution studies of microbial dynamics in complex environments.

Multipartite Fluorogenic Sensors for Monitoring Tyrosine Phosphatase Activity.

Fluorogenic substrates are essential tools for studying the activity of many enzymes including the protein tyrosine phosphatases (PTPs). Here, we have taken the first step toward the development of genetically encodable sensors for PTP activity using fluorescent and fluorogen-activating proteins. The Fluorescence-Activating and absorption Shifting Tag (FAST) is a small protein that becomes fluorescent upon binding to a small molecule dye. We demonstrate that FAST protein can be used as a sensor for PTP-mediated dephosphorylation of phosphorylated dye molecules. Phosphorylated 4-hydroxybenzylidene rhodanine (pHBR) is not able to bind to the FAST protein and induce fluorescence, but provides a sensitive assay for PTP activity, readily detecting 100 pM concentrations of PTP1B in the presence of FAST with a kcat value of 19±1 s-1 and a KM value of 93±3 μM. In addition, while phosphorylation of the C-terminal peptide of split GFP does not result in appreciable change in fluorescence of the reconstituted protein, phosphorylation of the C-terminal peptide of the split FAST protein abrogates fluorescence. Upon PTP-mediated dephosphorylation of the C-terminal peptide, the ability of the N- and C-terminal components to form a fluorescent complex with the small molecule dye is restored, leading to fluorescence.

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants

In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using 3-Hydroxypyridin-4(1H)-one-Fluorescein Probes.

The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.

GFP and FAP imaging of neuropeptide release in Drosophila.

Genetics in Drosophila have revealed the role of neuropeptides in development and behavior. However, determining when and where neuropeptides are released has been challenging. Furthermore, the cell biology underlying neuropeptide release has largely been unexplored. Thus, it has not been possible to determine whether changes in neuropeptide immunofluorescence reflect traffic and/or release, and in neurons where such changes are not detectable, conclusions about neuropeptide release have been formulated based on the assumption that electrical and Ca2+ recordings are accurate and quantitative predictors of release. Recently, the advent of optical detection of neuropeptides tagged with fluorescent proteins and fluorogen-activating proteins (FAPs) has made it feasible to directly image vesicle traffic and exocytosis that mediates neuropeptide release in peripheral synapses and in the brain. In fact, these approaches have led to the discovery of unexpected insights concerning neuropeptide release. Here procedures are presented for optimizing fluorescence imaging of neuropeptides tagged with green fluorescent protein or a FAP.

Imaging Neuropeptide Release from Drosophila Clock and Motor Neurons.

Electrophysiological studies of synaptic function do not robustly report release of neuropeptides and neurotrophins. These limitations have been overcome with the presynaptic expression of optical release reporters based on green fluorescent protein and fluorogen-activating protein. Here we describe how to image neuropeptide release in Drosophila at the neuromuscular junction and in the adult brain.

A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.

Symmetry-breaking malachite green as a near-infrared light-activated fluorogenic photosensitizer for RNA proximity labeling.

Cellular RNA is asymmetrically distributed in cells and the regulation of RNA localization is crucial for proper cellular functions. However, limited chemical tools are available to capture dynamic RNA localization in complex biological systems with high spatiotemporal resolution. Here, we developed a new method for RNA proximity labeling activated by near-infrared (NIR) light, which holds the potential for deep penetration. Our method, termed FAP-seq, utilizes a genetically encoded fluorogen activating protein (FAP) that selectively binds to a set of substrates known as malachite green (MG). FAP binding restricts the rotation of MG and rapidly activates its fluorescence in a wash-free manner. By introducing a monoiodo modification to MG, we created a photosensitizer (MG-HI) with the highest singlet oxygen generation ability among various MG derivatives, enabling both protein and RNA proximity labeling in live cells. New insights are provided inthe transcriptome analysis with FAP-seq, while a deeper understanding of the symmetry-breaking structural arrangement of FAP-MG-HI was obtained through molecular dynamics simulations. Overall, our wash-free and NIR light-inducible RNA proximity labeling method (FAP-seq) offers a powerful and versatile approach for investigating complex mechanisms underlying RNA-related biological processes.

Ca2+ and cAMP open differentially dilating synaptic fusion pores.

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. Here, we used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then show that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP).

NanoLuc Luciferase as a Fluorogen-Activating Protein for GFP Chromophore Based Fluorogens.

In this work, we showed that the well-known NanoLuc luciferase can act as a fluorogen activating protein for various arylidene-imidazolones structurally similar to the Kaede protein chromophore. We showed that such compounds can be used as fluorescent sensors for this protein and can also be used in pairs with it in fluorescent microscopy as a genetically encoded tag.

Insights into the Photodynamics of Fluorescence Emission and Singlet Oxygen Generation of Fluorogen Activating Protein-Malachite Green Systems.

The self-assembled fluorogen activating protein (FAP)-malachite green (MG) complex is a well-established protein-ligand system, which can realize binding-caused fluorescence turn-on of MG and singlet oxygen (1 O2 ) generation by MG iodination. To clarify the mechanism of fluorescence activation and 1 O2 generation, the photodynamics of different halogen-substituted MG derivatives and their corresponding FAP-MG complexes were studied by femtosecond transient absorption spectroscopy and theoretical computations. The results show that the rotation of MG is restricted by FAP binding, which prevents a rapid internal conversion to allow a longer lifetime for the excited MG to undergo fluorescence emission and intersystem crossing. Moreover, these FAP-MG complexes exhibit notably varied fluorescence quantum yields (ΦFL ) and 1 O2 yields. The study on the decay pathways indicates that such an anti-heavy atom effect predominately stems from the lifetimes of the excited-state species. The photodynamic mechanism study here will lead to more advanced FAP-MG systems with high spatiotemporal resolution.

Photodynamic therapy-improved oncolytic bacterial immunotherapy with FAP-encoding S. typhimurium.

Genetically engineered bacterial cancer therapy presents several advantages over conventional therapies. However, the anticancer effects of bacterium-based therapies remain insufficient, and serious side effects may be incurred with the increase in therapeutic dosages. Photodynamic therapy (PDT) suppresses tumor growth by producing reactive oxygen species (ROS) through specific laser-activated photosensitizers (PSs). Tumor-specific PS delivery and activatable ROS generation are two critical aspects for PDT advancement. Here, we propose PDT-enhanced oncolytic bacterial immunotherapy (OBI) by using genetically engineered avirulent Salmonella expressing a fluorogen-activating protein (FAP). Upon binding to a fluorogen, FAP could be activated and generate fluorescence and ROS. The instant activation of persistent fluorescence was detected in tumors through bacterium-based imaging. In a colon cancer model, PDT-OBI showed an enhanced tumor inhibition effect and prolonged animal survival. Mechanically, PDT generated ROS, resulting in the killing of cancer cells and over-accumulated bacteria. The pathogen-associated molecular patterns and damage-associated molecular patterns released from the destroyed bacteria and cancer cells recruited and activated immune cells (macrophages, neutrophils, and dendritic cells), which released additional proinflammatory cytokines (TNF-α and IL-1β); reduced anti-inflammatory cytokines (IL-10); and further enhanced immune cell infiltration in a positive-feedback manner, thus reducing bacterium-induced side effects and improving anticancer activities. This synergistic therapy has promising potential for cancer immunotherapy.

Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins.

A need in synthetic biology is the ability to precisely and efficiently make flexible fully designed vectors that addresses challenging cloning strategies of single plasmids that rely on combinatorial co-expression of a multitude of target and bait fusion reporters useful in projects like library screens. For these strategies, the regulatory elements and functional components need to correspond perfectly to project specific sequence elements that facilitate easy exchange of these elements. This requires systematic implementation and building on recent improvements in Golden Gate (GG) that ensures high cloning efficiency for such complex vectors. Currently, this is not addressed in the variety of molecular GG cloning techniques in synthetic biology. Here, we present the bottom-up design and plasmid synthesis to prepare 10 kb functional yeast secrete and display plasmids that uses an optimized version of GG in combination with fluorogen-activating protein reporter technology. This allowed us to demonstrate nanobody/target protein interactions in a single cell, as detected by cell surface retention of secreted target proteins by cognate nanobodies. This validates the GG constructional approach and suggests a new approach for discovering protein interactions. Our GG assembly platform paves the way for vector-based library screening and can be used for other recombinant GG platforms.

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2.

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

“Fluorescence-Activating and absorption-Shifting Tag” (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization.

Add and Go: FRET Acceptor for Live-Cell Measurements Modulated by Externally Provided Ligand.

A substantial number of genetically encoded fluorescent sensors rely on the changes in FRET efficiency between fluorescent cores, measured in ratiometric mode, with acceptor photobleaching or by changes in fluorescence lifetime. We report on a modulated FRET acceptor allowing for simplified one-channel FRET measurement based on a previously reported fluorogen-activating protein, DiB1. Upon the addition of the cell-permeable chromophore, the fluorescence of the donor-fluorescent protein mNeonGreen decreases, allowing for a simplified one-channel FRET measurement. The reported chemically modulated FRET acceptor is compatible with live-cell experiments and allows for prolonged time-lapse experiments with dynamic energy transfer evaluation.

Fluorogen-Activating-Protein-Loaded Tantalum Oxide Nanoshells for in Vivo On-Demand Fluorescence/Photoacoustic Imaging.

Optical imaging of targeted compartments within living animals has been widely adopted in many research areas. In particular, various fluorescence-based probes and emerged photoacoustic molecules that enable sensitive and specific imaging through tissue have greatly advanced clinically relevant studies. However, delivery and signal penetration have placed requirements on the performance of conventional optical probes. Here, we use hallow tantalum oxide (TaOx) nanoparticles to enclose fluorogen-activating protein (FAP) for the in vivo fluorescence and photoacoustic imaging of cancer cells. We found that the TaOx shell can provide a natural cover for the enclosed fluorogen/FAP complexes, protecting them from photobleaching and common biodegradation. Moreover, we have developed a near-infrared excitable tetrafluorinated photoacoustic fluorogen for the specific and persistent photoacoustic imaging of tumors. We believe that this enclosing and delivery strategy of optical biomolecules will be an attractive alternative for bioimaging.

Computational redesign of a fluorogen activating protein with Rosetta.

The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of in vitro screening and selection. Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed.