Kinetic isotope effect reveals rate-limiting step in green-to-red photoconvertible fluorescent proteins.

Photoconvertible fluorescent proteins (pcFPs) undergo a slow photochemical transformation when irradiated with blue light. Since their emission is shifted from green to red, pcFPs serve as convenient fusion tags in several cutting-edge biological imaging technologies. Here, a pcFP termed the Least Evolved Ancestor (LEA) was used as a model system to determine the rate-limiting step of photoconversion. Perdeuterated histidine residues were introduced by isotopic enrichment and chromophore content was monitored by absorbance. pH-dependent photoconversion experiments were carried out by exposure to 405-nm light followed by dark equilibration. The loss of green chromophore correlated well with the rise of red, and maximum photoconversion rates were observed at pH 6.5 (0.059 ± 0.001 min-1 for red color acquisition). The loss of green and the rise of red provided deuterium kinetic isotope effects (DKIEs) that were identical within error, 2.9 ± 0.9 and 3.8 ± 0.6, respectively. These data indicate that there is one rate-determining step in the light reactions of photoconversion, and that CH bond cleavage occurs in the transition state of this step. We propose that these reactions are rate-limited on the min time scale by the abstraction of a proton at the His62 beta-carbon. A conformational intermediate such as a twisted or isomerized chromophore is proposed to slowly equilibrate in the dark to generate the red form. Additionally, His62 may shuttle protons to activate Glu211 to serve as a general base, while also facilitating beta-elimination. This idea is supported by a recent X-ray structure of methylated His62.

Viscosity-Induced Emission of 5-(Diarylmethylene)imidazolone with Extended Conjugation via Attachment of N-Methylpyrrole at the 2-Position.

We have developed a series of 2-monoaryl-5-diarylmethylene analogs of the green fluorescent protein chromophore to study their viscosity-induced emission (VIE) properties. The analogs were synthesized by a condensation with methyl imidate and N-(diarylmethylene)glycinate. Among the analogs, the N-methylpyrrol-2-yl-substituted analog 1h induced the most remarkable VIE behavior in triglyceride and lipid bilayers probably due to the high π-electron-rich property of the pyrrole ring. The pyrrole substituent in imidazolone analogs can be expected to become a common template for introducing VIE behavior.

Sequence-developability mapping of affibody and fibronectin paratopes via library-scale variant characterization.

Protein developability is requisite for use in therapeutic, diagnostic, or industrial applications. Many developability assays are low throughput, which limits their utility to the later stages of protein discovery and evolution. Recent approaches enable experimental or computational assessment of many more variants, yet the breadth of applicability across protein families and developability metrics is uncertain. Here, three library-scale assays-on-yeast protease, split green fluorescent protein (GFP), and non-specific binding-were evaluated for their ability to predict two key developability outcomes (thermal stability and recombinant expression) for the small protein scaffolds affibody and fibronectin. The assays' predictive capabilities were assessed via both linear correlation and machine learning models trained on the library-scale assay data. The on-yeast protease assay is highly predictive of thermal stability for both scaffolds, and the split-GFP assay is informative of affibody thermal stability and expression. The library-scale data was used to map sequence-developability landscapes for affibody and fibronectin binding paratopes, which guides future design of variants and libraries.

Preparation of Chitosan Nanoparticles through a Readily Solvent-Exchange Process for Efficient and Enhanced Gene Delivery.

In view of the excellent prospects of gene therapy and the potential safety and immunogenicity issues challenged by viral vectors, it is of great significance to develop a nonviral vector with low toxicity and low cost. In this work, we report a chitosan nanoparticle (CSNP) to be used as a gene vector prepared through a facile solvent-exchange strategy. Chitosan is first dissolved in ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIM Ac), and then, the solvent is exchanged with water/phosphate-buffered saline (PBS) to remove ionic liquid, forming a final CSNP dispersion after ultrasonication. The prepared CSNP shows a positive surface charge and can condense green fluorescent protein-encoding plasmid (pGFP) at weight ratios (CSNP/pGFP) of 5/1 or higher. Dynamic light scattering size and ζ-potential characterization and gel retardation results confirm the formation of CSNP/pGFP complexes. Compared with plain pGFP, efficient cellular internalization and significantly enhanced green fluorescent protein (GFP) expression are observed by using CSNP as a plasmid vector. Benefitting from the intrinsic biocompatibility, low cost, low immunogenicity, and abundant sources of chitosan, as well as the facile preparation and the efficient gene transfection capacity of CSNP, it is believed that this CSNP could be used as a nonviral gene vector with great clinical translational potentials.

Characterization of peptide-fused protein assemblies in living cells.

Assembly of de novo peptides designed from scratch is in a semi-rational manner and creates artificial supramolecular structures with unique properties. Considering that the functions of various proteins in living cells are highly regulated by their assemblies, building artificial assemblies within cells holds the potential to simulate the functions of natural protein assemblies and engineer cellular activities for controlled manipulation. How can we evaluate the self-assembly of designed peptides in cells? The most effective approach involves the genetic fusion of fluorescent proteins (FPs). Expressing a self-assembling peptide fused with an FP within cells allows for evaluating assemblies through fluorescence signal. When µm-scale assemblies such as condensates are formed, the peptide assemblies can be directly observed by imaging. For sub-µm-scale assemblies, fluorescence correlation spectroscopy analysis is more practical. Additionally, the fluorescence resonance energy transfer (FRET) signal between FPs is valuable evidence of proximity. The decrease in fluorescence anisotropy associated with homo-FRET reveals the properties of self-assembly. Furthermore, by combining two FPs, one acting as a donor and the other as an acceptor, the heteromeric interaction between two different components can be studied through the FRET signal. In this chapter, we provide detailed protocols, from designing and constructing plasmid DNA expressing the peptide-fused protein to analysis of self-assembly in living cells.

Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

One-Step Purification and N-Terminal Functionalization of Bioactive Proteins via Atypically Split Inteins.

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (ß-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.

Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis.

Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.

Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions.

A viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with 1000 localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long time scales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the entire life cycle of a single virus.

Sortase A-Based Post-translational Modifications on Encapsulin Nanocompartments.

Protein-based encapsulin nanocompartments, known for their well-defined structures and versatile functionalities, present promising opportunities in the fields of biotechnology and nanomedicine. In this investigation, we effectively developed a sortase A-mediated protein ligation system in Escherichia coli to site-specifically attach target proteins to encapsulin, both internally and on its surfaces without any further in vitro steps. We explored the potential applications of fusing sortase enzyme and a protease for post-translational ligation of encapsulin to a green fluorescent protein and anti-CD3 scFv. Our results demonstrated that this system could attach other proteins to the nanoparticles' exterior surfaces without adversely affecting their folding and assembly processes. Additionally, this system enabled the attachment of proteins inside encapsulins which varied shapes and sizes of the nanoparticles due to cargo overload. This research developed an alternative enzymatic ligation method for engineering encapsulin nanoparticles to facilitate the conjugation process.

Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy.

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

Objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) for studying the fusion dynamics of protein phase separation.

Protein phase separation plays a very important role in many biological processes and is closely related to the occurrence and development of some serious diseases. So far, the fluorescence imaging method and fluorescence correlation spectroscopy (FCS) have been frequently used to study the phase separation behavior of proteins. Due to the wide size distribution of protein condensates in phase separation from nano-scale to micro-scale in solution and living cells, it is difficult for the fluorescence imaging method and conventional FCS to fully reflect the real state of protein phase separation in the solution due to the low spatio-temporal resolution of the conventional fluorescence imaging method and the limited detection area of FCS. Here, we proposed a novel method for studying the protein phase separation process by objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS). In this study, CRDBP proteins were used as a model and respectively fused with fluorescent proteins (EGFP and mCherry). We first compared conventional FCS and Scan-FCS methods for characterizing the CRDBP protein phase separation behaviors and found that the reproducibility of Scan-FCS is significantly improved by the scanning mode. We studied the self-fusion process of mCherry-CRDBP and EGFP-CRDBP and observed that the phase change concentration of CRDBP was 25 nM and the fusion of mCherry-CRDBP and EGFP-CRDBP at 500 nM was completed within 70 min. We studied the effects of salt concentration and molecular crowding agents on the phase separation of CRDBP and found that salt can prevent the self-fusion of CRDBP and molecular crowding agents can improve the self-fusion of CRDBP. Furthermore, we found the recruitment behavior of CRDBP to ß-catenin proteins and studied their recruitment dynamics. Compared to conventional FCS, Scan-FCCS can significantly improve the reproducibility of measurements due to the dramatic increase of detection zone, and more importantly, this method can provide information about self-fusion and recruitment dynamics in protein phase separation.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.

Unveiling success determinants for AMB-assisted phase expansion of fusion proteins in ARP/wARP.

Fusion proteins (FPs) are frequently utilized as a biotechnological tool in the determination of macromolecular structures using X-ray methods. Here, we explore the use of different protein tags in various FP, to obtain initial phases by using them in a partial molecular replacement (MR) and constructing the remaining FP structure with ARP/wARP. Usually, the tag is removed prior to crystallization, however leaving the tag on may facilitate crystal formation, and structural determination by expanding phases from known to unknown segments of the complex. In this study, the Protein Data Bank was mined for an up-to-date list of FPs with the most used protein tags, Maltose Binding Protein (MBP), Green Fluorescent Protein (GFP), Thioredoxin (TRX), Glutathione transferase (GST) and the Small Ubiquitin-like Modifier Protein (SUMO). Partial MR using the protein tag, followed by automatic model building, was tested on a subset of 116 FP. The efficiency of this method was analyzed and factors that influence the coordinate construction of a substantial portions of the fused protein were identified. Using MBP, GFP, and SUMO as phase generators it was possible to build at least 75 % of the protein of interest in 36 of the 116 cases tested. Our results reveal that tag selection has a significant impact; tags with greater structural stability, such as GFP, increase the success rate. Further statistical analysis identifies that resolution, Wilson B factor, solvent percentage, completeness, multiplicity, protein tag percentage in the FP (considering amino acids), and the linker length play pivotal roles using our approach. In cases where a structural homologous is absent, this method merits inclusion in the toolkit of protein crystallographers.

A GFP Inspired 8-Methoxyquinoline-Derived Fluorescent Molecular Sensor for the Detection of Zn2+ by Two-Photon Microscopy.

An effective, GFP-inspired fluorescent Zn2+ sensor is developed for two-photon microscopy and related biological application that features an 8-methoxyquinoline moiety. Excellent photophysical characteristics including a 37-fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two-photon cross-section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn-on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc-bound compounds are presented.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

A novel method for high level production of protein glutaminase by sfGFP tag in Bacillus subtilis.

Protein glutaminase (PG; EC 3.5.1.44) is a novel deamidase that helps to improve functional properties of food proteins. Currently, the highest activated PG enzyme activity was 26 U/mg when recombinantly expressed via the twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum. In this study, superfolder green fluorescent protein (sfGFP) was used to replace traditional signal peptides to facilitate efficient heterologous expression and secretion of Propeptide-Protein glutaminase (PP) in Bacillus subtilis. The fusion protein, sfGFP-PP, was secreted from 12 h of fermentation and reached its highest extracellular expression at 28 h, with a secretion efficiency of about 93 %. Moreover, when fusing sfGFP with PP at the N-terminus, it significantly enhances PG expression up to 26 U/mL by approximately 2.2-fold compared to conventional signal-peptides- guided PP with 11.9 U/mL. Finally, the PG enzyme activity increased from 26 U/mL to 36.9 U/mL after promoter and RBS optimization. This strategy not only provides a new approach to increase PG production as well as extracellular secretion but also offers sfGFP as an effective N-terminal tag for increased secreted production of difficult-to-express proteins.

Influence of Fluorescence Lifetime Selections and Conformational Flexibility on Brightness of FusionRed Variants.

Fluorescent proteins (FPs) for bioimaging are typically developed by screening mutant libraries for clones with improved photophysical properties. This approach has resulted in FPs with high brightness, but the mechanistic origins of the improvements are often unclear. We focused on improving the molecular brightness in the FusionRed family of FPs with fluorescence lifetime selections on targeted libraries, with the aim of reducing nonradiative decay rates. Our new variants show fluorescence quantum yields of up to 75% and lifetimes >3.5 ns. We present a comprehensive analysis of these new FPs, including trends in spectral shifts, photophysical data, photostability, and cellular brightness resulting from codon optimization. We also performed all-atom molecular dynamics simulations to investigate the impact of side chain mutations. The trajectories reveal that individual mutations reduce the flexibility of the chromophore and side chains, leading to an overall reduction in nonradiative rates.

Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy.

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 µm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1-2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.