A hybrid pathway for self-sustained luminescence.

The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.

Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

An improved pathway for autonomous bioluminescence imaging in eukaryotes.

The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.

Meta-CF3-Substituted Analogues of the GFP Chromophore with Remarkable Solvatochromism.

In this work, we have shown that the introduction of a trifluoromethyl group into the me-ta-position of arylidene imidazolones (GFP chromophore core) leads to a dramatic increase in their fluorescence in nonpolar and aprotic media. The presence of a pronounced solvent-dependent gradation of fluorescence intensity makes it possible to use these substances as fluorescent polarity sensors. In particular, we showed that one of the created compounds could be used for selective labeling of the endoplasmic reticulum of living cells.

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.

Domain Truncation in Hispidin Synthase Orthologs from Non-Bioluminescent Fungi Does Not Lead to Hispidin Biosynthesis.

Hispidin is a polyketide found in plants and fungi. In bioluminescent fungi, hispidin serves as a precursor of luciferin and is produced by hispidin synthases. Previous studies revealed that hispidin synthases differ in orthologous polyketide synthases from non-bioluminescent fungi by the absence of two domains with predicted ketoreductase and dehydratase activities. Here, we investigated the hypothesis that the loss of these domains in evolution led to the production of hispidin and the emergence of bioluminescence. We cloned three orthologous polyketide synthases from non-bioluminescent fungi, as well as their truncated variants, and assessed their ability to produce hispidin in a bioluminescence assay in yeast. Interestingly, expression of the full-length enzyme hsPKS resulted in dim luminescence, indicating that small amounts of hispidin are likely being produced as side products of the main reaction. Deletion of the ketoreductase and dehydratase domains resulted in no luminescence. Thus, domain truncation by itself does not appear to be a sufficient step for the emergence of efficient hispidin synthases from orthologous polyketide synthases. At the same time, the production of small amounts of hispidin or related compounds by full-length enzymes suggests that ancestral fungal species were well-positioned for the evolution of bioluminescence.

Systematic Comparison of Plant Promoters in Nicotiana spp. Expression Systems.

We report a systematic comparison of 19 plant promoters and 20 promoter-terminator combinations in two expression systems: agroinfiltration in Nicotiana benthamiana leaves, and Nicotiana tabacum BY-2 plant cell packs. The set of promoters tested comprised those not present in previously published work, including several computationally predicted synthetic promoters validated here for the first time. The expression of EGFP driven by different promoters varied by more than two orders of magnitude and was largely consistent between two tested Nicotiana systems. We confirmed previous reports of significant modulation of expression by terminators, as well as synergistic effects of promoters and terminators. Additionally, we observed non-linear effects of gene dosage on expression level. The dataset presented here can inform the design of genetic constructs for plant engineering and transient expression assays.

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.

Heterogeneity of the GFP fitness landscape and data-driven protein design.

Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design - instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering.

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.

Transient Fluorescence Labeling: Low Affinity-High Benefits.

Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them-the requirement for high photostability-can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.

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.

Treacle and TOPBP1 control replication stress response in the nucleolus.

Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress-response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle-TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle-TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.

Chromophore reduction plus reversible photobleaching: how the mKate2 "photoconversion" works.

mKate red-to-green photoconversion is a non-canonical type of phototransformation in fluorescent proteins, with a poorly understood mechanism. We have hypothesized that the daughter mKate2 protein may also be photoconvertible, and that this phenomenon would be connected with mKate(2) chromophore photoreduction. Indeed, upon the intense irradiation of the protein sample supplemented by sodium dithionite, the accumulation of green as well as blue spectral forms is enhanced. The reaction was shown to be reversible upon the reductant's removal. However, an analysis of the fluorescence microscopy data, absorption spectra, kinetics and time-resolved fluorescence spectroscopy revealed that the short-wavelength spectral forms of mKate(2) exist before photoactivation, that their fractions increase light-independently after dithionite addition, and that the conversion is facilitated by the photobleaching of the red chromophore form.

Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein-Chromophore Interactions.

Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M-1 cm-1 , is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein-fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions.

NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells.

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein.

Using benzylidene imidazolone core, we created a panel of color-shifted fluorogenic ligands for FAST protein without compromise to the binding efficiency and the utility for live-cell protein labeling. This study highlights the potential of benzylidene imidazolones derivatives for rapid expansion of a pallet of live-cell fluorogenic labeling tools.

A General Mechanism of Green-to-Red Photoconversions of GFP.

Here we dissect the phenomena of oxidative and reductive green-to-red photoconversion of the Green Fluorescent Protein. We characterize distinct orange- and red-emitting forms (λabs/λem = 490/565 nm; λabs/λem = 535/600 nm) arising during the Enhanced Green Fluorescent Protein (EGFP) photoconversion under low-oxygen conditions in the presence of reductants. These forms spectroscopically differ from that observed previously in oxidative redding (λabs/λem = 575/607 nm). We also report on a new green-emitting state (λabs/λem = 405/525 nm), which is formed upon photoconversion under the low-oxygen conditions. Based on the spectral properties of these forms, their light-independent time evolution, and the high-level computational studies, we provide a structural basis for various photoproducts. Under the low-oxygen conditions, the neutral quinoid-like structure formed via a two-electron oxidation process is found to be a key intermediate and a most likely candidate for the novel green-emitting state of the chromophore. The observed large Stokes shift is traced to the formation of the zwitterionic form of the chromophore in the excited state. Subsequently, this form undergoes two types of cyclization reactions, resulting in the formation of either the orange-emitting state (λabs/λem = 490/565 nm) or the red-emitting form (λabs/λem = 535/600 nm). The T65G mutant lacks one of the proposed cyclization pathways and, indeed, the photoconverted T65G EGFP exhibits a single orange-emitting state. In oxidative redding, the red-emitting state resembles the structure of the chromophore from asFP595 (λabs/λem = 572/595 nm), which is directly formed upon two-electron oxidation and deprotonation bypassing the formation of the quinoid-like structure. Our results disclose a general "oxidative" mechanism of various green-to-red photoconversions of EGFP, providing a link between oxidative redding and the photoconversion under low-oxygen conditions.

Structure-Based Rational Design of Two Enhanced Bacterial Lipocalin Blc Tags for Protein-PAINT Super-resolution Microscopy.

Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes. Once photobleached, the dye in the complex is replaced with a fresh fluorogen available in the sample. With suitable kinetics, this replacement creates fluorescence blinking required for attaining super-resolution and overcomes photobleaching associated with the loss of an irreplaceable fluorophore. Here we report on the rational design of two protein-PAINT tags based on the 1.58 Å crystal structure of the DiB1:M739 complex, an improved green-emitting DiB3/F74V:M739 and a new orange-emitting DiB3/F53L:M739. They outperform previously reported DiB-based tags to become best in class biomarkers for protein-PAINT. The new tags advance protein-PAINT from the proof-of-concept to a reliable tool suitable for prolonged super-resolution imaging of intracellular proteins in fixed and living cells and two-color PAINT-like nanoscopy with a single fluorogen.