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Undergraduate laboratory courses are essential to teaching core principles in STEM. This course, Quantitative Biological Methods, provides a unique approach to teaching molecular biology research techniques to students, in a laboratory that is delivered in a sequence that parallels standard biomedical research laboratory protocols. Students attend a lecture where they are taught the essential principles of biomedical research, and a lab where they learn to use laboratory equipment, perform experiments, and purify and quantify DNA and proteins. The course begins with an introduction to laboratory safety, pipetting, centrifugation, spectrophotometry, and other basic laboratory techniques. Next, the lab focuses on the purification and analysis of glutathione S-transferase (GST) fused to green fluorescent protein (GFP) from an Escherichia coli lysate. Students study this GST-GFP fusion protein and perform protein quantification, enzyme assays, chromatography, fluorescent detection, normalization, SDS-PAGE, and western blotting. Students then learn recombinant DNA technology using the GST-GFP vector that was the source of the fusion protein in the prior labs, and perform ligation, transformation of E. coli cells, blue/white screening, DNA purification via a miniprep, PCR, DNA quantification, restriction enzyme digestion, and agarose gel electrophoresis. Students write laboratory reports to demonstrate an understanding of the principles of the laboratory methods, and they must present and critically analyze their data. The lab methods described herein aim to emphasize the core molecular biology principles and techniques, prepare students for work in a biomedical research laboratory, and introduce students to both GST and GFP, two versatile laboratory proteins., (© 2021 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Catalytically active materials for the enhancement of personalized protective equipment (PPE) could be advantageous to help alleviate threats posed by neurotoxic organophosphorus compounds (OPs). Accordingly, a chimeric protein comprised of a supercharged green fluorescent protein (scGFP) and phosphotriesterase from Agrobacterium radiobacter (arPTE) was designed to drive the polymer surfactant (S - )-mediated self-assembly of microclusters to produce robust, enzymatically active materials. The chimera scGFP-arPTE was structurally characterized via circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering, and its biophysical properties were determined. Significantly, the chimera exhibited greater thermal stability than the native constituent proteins, as well as a higher catalytic turnover number ( k cat ). Furthermore, scGFP-arPTE was electrostatically complexed with monomeric S - , driving self-assembly into [scGFP-arPTE][S - ] nanoclusters, which could be dehydrated and cross-linked to yield enzymatically active [scGFP-arPTE][S - ] porous films with a high-order structure. Moreover, these clusters could self-assemble within cotton fibers to generate active composite textiles without the need for the pretreatment of the fabrics. Significantly, the resulting materials maintained the biophysical activities of both constituent proteins and displayed recyclable and persistent activity against the nerve agent simulant paraoxon.

Bioimaging techniques require development of a wide variety of fluorescent probes that absorb and emit red light. One way to shift absorption and emission of a chromophore to longer wavelengths is to modify its chemical structure by adding polycyclic aromatic hydrocarbon (PAH) fragments, thus increasing the conjugation length of a molecule while maintaining its rigidity. Here, we consider four novel classes of conformationally locked Green Fluorescent Protein (GFP) chromophore derivatives obtained by extending their aromatic systems in different directions. Using high-level ab initio quantum chemistry calculations, we show that the alteration of their electronic structure upon annulation may unexpectedly result in a drastic change of their fluorescent properties. A flip of optically bright and dark electronic states is most prominent in the symmetric fluorene-based derivative. The presence of a completely dark lowest-lying excited state is supported by the experimentally measured extremely low fluorescence quantum yield of the newly synthesized compound. Importantly, one of the asymmetric modes of annulation provides a very promising strategy for developing red-shifted molecular emitters with an absorption wavelength of ∼600 nm, having no significant impact on the character of the bright S-S 1 transition.

Green fluorescent protein (GFP)-like fluorescent proteins have been found in more than 120 species. Although the proteins have little sequence identity, Gly31, 33, and 35 are 87, 100, and 95% conserved across all species, respectively. All GFP-like proteins have a β-barrel structure composed of 11 β-sheets, and the 3 conserved glycines are located in the second β-sheet. Molecular dynamics (MD) simulations have shown that mutating one or more of the glycines to alanines most likely does not reduce chromophore formation in correctly folded immature fluorescent proteins. MD and protein characterization of alanine mutants indicate that mutation of the conserved glycines leads to misfolding. Gly31, 33, and 35 are essential to maintain the integrity of the β 1-3 triad that is the last structural element to slot in place in the formation of the canonical fluorescent protein β-barrel. Glycines located in β-sheets may have a similar role in the formation of other non-GFP β-barrels.

Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B 42-94 ) for in vitro studies. First, we expressed fluorescently tagged BCL11B 42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B 42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue., (© 2021. The Author(s).)

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet-state quenchers or redox-active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the β-barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Green fluorescent proteins (GFP) are commonly used as fluorescent tags and biosensors in cell biology and medicine. However, the propensity of GFP-like proteins to aggregate and the consequence of intermolecular interaction for their application have not been thoroughly examined. In this work, alternative aggregation pathways of superfolder green fluorescent protein (sfGFP) were demonstrated using a spectroscopic and microscopic study of the samples prepared by equilibrium microdialysis. Besides oligomerization of native monomers, we showed for the first time the condition-specific formation by sfGFP of either amyloid fibrils (at increased temperature or acidity) or amorphous aggregates (at physiological conditions). Both types of sfGFP aggregates had lost green fluorescence and were toxic to cells. Thus, when using GFP-like proteins as fluorescent tags, one should take into account their high ability to form aggregates with lost unique visible fluorescence in the cellular environment, which affects cell viability. Moreover, the results of this work cast doubt on the correctness of the data on the fibrillogenesis of various amyloidogenic proteins obtained using their fusion with GFP-like proteins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fluorescent proteins (FP) are an integral part of modern biology due to its diverse biochemical and photophysical properties. The boundaries of FP have been extended through conventional mutagenesis and directed evolution approaches. Engineering of FP based on the standard genetic code consisting of 20 amino acids with limited functional groups restrict its diversification. Degeneracy of genetic code has helped in covering this substantial gap through genetic code engineering, wherein introduction of unnatural amino acid (UAA) analogues resulted in a collection of FP with varying properties. This review features the work carried till date in the area of FP incorporated with UAAs and explores strategies employed for incorporation, impact of UAAs in chromophore and surrounding residues and changes in inherent properties of FP. The long-standing association of FP as a tool for high throughput screening of orthogonal aaRS/tRNA pairs used in site specific incorporation of UAAs is expounded. Insertion of UAAs in FP has enabled their use in contemporary fields such as biophotovoltaics, bioremediation, biosensors, biomaterials and imaging of acidic vesicles. Thus, expansion of genetic code of FP is envisaged to rejig the existing spectra of colors and future research initiative in this direction is expected to glow brighter and brighter., (Copyright © 2021 Elsevier B.V. All rights reserved.)

During injured tissue regeneration, the extracellular matrix plays a key role in controlling and coordinating various cellular events by binding and releasing secreted proteins in addition to promoting cell adhesion. Herein, we develop a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A as an artificial extracellular matrix for regenerative therapy. We show that the jigsaw-shaped self-assembling peptide forms several-micrometer-long supramolecular nanofibers through a helix-to-strand transition to afford a hydrogel under physiological conditions and disperses homogeneously in the hydrogel. The molecular- and macro-scale supramolecular properties of the jigsaw-shaped self-assembling peptide hydrogel allow efficient incorporation and sustained release of vascular endothelial growth factor, and demonstrate cell transplantation-free regenerative therapeutic effects in a subacute-chronic phase mouse stroke model. This research highlights a therapeutic strategy for injured tissue regeneration using the jigsaw-shaped self-assembling peptide supramolecular hydrogel., (© 2021. The Author(s).)

Bilin-binding fluorescent proteins like UnaG-bilirubin are noncovalent ligand-dependent reporters for oxygen-free microscopy but are restricted to blue and far-red fluorescence. Here we describe a high-throughput screening approach to provide a new UnaG-ligand pair that can be excited in the 532 nm green excitation microscopy channel. We identified a novel orange UnaG-ligand pair that maximally emits at 581 nm. Whereas the benzothiazole-based ligand itself is nominally fluorescent, the compound binds UnaG with high affinity ( K d = 3 nM) to induce a 2.5-fold fluorescence intensity enhancement and a 10 nm red shift. We demonstrated this pair in the anaerobic fluorescence microscopy of the prevalent gut bacterium Bacteroides thetaiotaomicron and in Escherichia coli . This UnaG-ligand pair can also be coupled to IFP2.0-biliverdin to differentiate cells in mixed-species two-color imaging. Our results demonstrate the versatility of the UnaG ligand-binding pocket and extend the ability to image cells at longer wavelengths in anoxic environments.

Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2 , with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m -carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.

Delivery of functional proteins into cells may help us understand how specific protein influences cell behavior as well as treat diseases caused by protein deficiency or loss-of-function mutations. However, protein cannot enter cells by diffusion. In this work, a novel cell biology tool for delivering recombinant proteins into mammalian cells was developed. We hijacked the intracellular transport routes used by the cholera toxin and took advantage of recent development on split intein that is compatible with denatured conditions and shows an exceptional splicing activity to deliver a protein of interest into mammalian cells. Here, we used green fluorescent protein and apoptin as proofs-of-concept. The results demonstrate that the cholera toxin B subunit alone could deliver other recombinant proteins into cells through either covalent conjugation or noncovalent interaction. Our method offers more than 10-fold better delivery efficiency than the tat cell-penetrating peptide and is selective for ganglioside-rich cells. This study adds a useful tool to the receptor-mediated intracellular targeting toolkit and opens possibility for the selective delivery of therapeutic proteins into ganglioside-rich cells.

Superresolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Superresolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photoconvertible fluorescent protein, respectively, resulting in the f ixation- r esistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and reactivated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.

Potassium acyltrifluoroborates (KATs) undergo chemoselective amide-forming ligations with hydroxylamines. Under aqueous, acidic conditions these ligations can proceed rapidly, with rate constants of ∼20 M -1 s -1 . The requirement for lower pH to obtain the fastest rates, however, limits their use with certain biomolecules and precludes in vivo applications. By mechanistic investigations into the KAT ligation, including kinetic studies, X-ray crystallography, and DFT calculations, we have identified a key role for a proton in accelerating the ligation. We applied this knowledge to the design and synthesis of 8-quinolyl acyltrifluoroborates, a new class of KATs that ligates with hydroxylamines at pH 7.4 with rate constants >4 M -1 s -1 . We trace the enhanced rate at physiological pH to unexpectedly high basicity of the 8-quinoline-KATs, which leads to their protonation even under neutral conditions. This proton assists the formation of the key tetrahedral intermediate and activates the leaving groups on the hydroxylamine toward a concerted 1,2-BF 3 shift that leads to the amide product. We demonstrate that the fast ligations at pH 7.4 can be carried out with a protein substrate at micromolar concentrations.

Strategies for investigating and optimizing the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations., (© 2021. The Author(s).)

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15 N-labeling of the C-terminal amide group of a single domain antibody (V HH ).

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78-7.7 μM) in vitro, but the activity was accompanied by pronounced cytotoxicity.

Immobilization of proteins on magnetic nanoparticles (MNPs) is an effective approach to improve protein stability and facilitate separation of immobilized proteins for repeated use. Herein, we exploited the efficient SpyTag-SpyCatcher chemistry for conjugation of functional proteins onto MNPs and established a robust magnetic-responsive nanoparticle platform for protein immobilization. To maximize the loading capacity and achieve outstanding water dispersity, the SpyTag peptide was incorporated into the surface-charged polymers of MNPs, which provided abundant active sites for Spy chemistry while maintaining excellent colloidal stability in buffer solution. Conjugation between enhanced green fluorescence protein (EGFP)-SpyCatcher-fused proteins and SpyTag-functionalized MNPs was efficient at ambient conditions without adding enzymes or chemical cross-linkers. Benefiting from the excellent water dispersity and interface compatibility, the surface Spy reaction has fast kinetics, which is comparable to that of the solution Spy reaction. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The immobilization process of EGFP on MNPs was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as bovine serum albumin and Tween 20. The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. In addition, experiments confirmed the retained magnetophoresis of the MNP after protein loading, demonstrating fast MNP recovery under an external magnetic field. This MNP is expected to provide a versatile and modular platform to achieve effective and specific immobilization of other functional proteins, enabling easy reuse and storage.

Super-resolution activity imaging maps the biochemical architecture of living cells yet currently overlooks the locations of collaborating regulators/effectors. Building on the fluorescence fluctuation increase by contact (FLINC) principle, here we devise Dronpa-chromophore-removed FLINC (DrFLINC), where the nonfluorescent Dronpa can nevertheless enhance TagRFP-T fluorescence fluctuations. Exploiting DrFLINC, we develop a superior red label and a next-generation activity sensor for context-rich super-resolution biosensing.

Motivated by the growing importance of single fluorescent protein biosensors (SFPBs) in biological research and the difficulty in rationally engineering these tools, we sought to increase the rate at which SFPB designs can be optimized. SFPBs generally consist of three components: a circularly permuted fluorescent protein, a ligand-binding domain, and linkers connecting the two domains. In the absence of predictive methods for biosensor engineering, most designs combining these three components will fail to produce allosteric coupling between ligand binding and fluorescence emission. While methods to construct diverse libraries with variation in the site of GFP insertion and linker sequences have been developed, the remaining bottleneck is the ability to test these libraries for functional biosensors. We address this challenge by applying a massively parallel assay termed "sort-seq," which combines binned fluorescence-activated cell sorting, next-generation sequencing, and maximum likelihood estimation to quantify the brightness and dynamic range for many biosensor variants in parallel. We applied this method to two common biosensor optimization tasks: the choice of insertion site and optimization of linker sequences. The sort-seq assay applied to a maltose-binding protein domain-insertion library not only identified previously described high-dynamic-range variants but also discovered new functional insertion sites with diverse properties. A sort-seq assay performed on a pyruvate biosensor linker library expressed in mammalian cell culture identified linker variants with substantially improved dynamic range. Machine learning models trained on the resulting data can predict dynamic range from linker sequences. This high-throughput approach will accelerate the design and optimization of SFPBs, expanding the biosensor toolbox.

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein-protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP., (© 2021. The Author(s).)

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging. Numerous spectroscopic studies of the isolated GFP chromophore have been aimed at understanding the electronic properties of GFP. Here, we build on earlier work [A. V. Bochenkova, C. Mooney, M. A. Parkes, J. Woodhouse, L. Zhang, R. Lewin, J. M. Ward, H. Hailes, L. H. Andersen and H. H. Fielding, Chem. Sci. , 2017, 8 , 3154] investigating the impact of fluorine and methoxy substituents that have been employed to tune the electronic structure of the GFP chromophore for use as fluorescent RNA tags. We present photoelectron spectra following photoexcitation over a broad range of wavelengths (364-230 nm) together with photoelectron angular distributions following photoexcitation at 364 nm, which are interpreted with the aid of quantum chemistry calculations. The results support the earlier high-level quantum chemistry calculations that predicted how fluorine and methoxy substituents tune the electronic structure and we find evidence to suggest that the methoxy substituents enhance internal conversion, most likely from the 2ππ* state which has predominantly Feshbach resonance character, to the 1ππ* state.

Isolation of nuclei tagged in specific cell types (INTACT) allows for stress-free and high-throughput analyses of cellular subpopulations. Here, we present an improved protocol for isolation of pure and high-quality GFP-labeled nuclei from frozen livers of INTACT mice, as well as protocols for downstream sequencing analyses. The adaptation to frozen tissue provides a pause point that allows sampling at multiple time points and/or phenotypic characterization of livers prior to nuclei isolation and downstream analyses. For complete details on the use of this protocol, please refer to Loft et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Genetically encoded molecular tools are crucial for live cell RNA imaging, and few are available for endogenous RNA imaging. We develop a new genetically encoded sensor using conformation switching RNA induced fluorogenic proteins that enable multicolor and signal-amplified imaging of endogenous RNAs. The sensor system is designed with an RNA sensing module and a degron-fused fluorescent protein reporter. Target RNA induces conformation switching of the RNA sensing module to form RNA aptamers that stabilize the degron-fused protein for fluorogenic imaging. This sensor is demonstrated for high-contrast imaging of survivin mRNA abundance and dynamics in live cells. Moreover, the sensor system is extended to a multicolor palette by screening fluorogenic proteins of distinct colors, and engineered into a signal amplifier using the split fluorescent protein design. The sensor is further exploited for imaging lncRNA MALAT-1 and its translocation dynamics during mitosis. Our sensor system can afford a valuable platform for RNA imaging in biomedical research and clinical theranostics.

The role of protonation states of the chromophore and its neighboring amino acid side chains of the reversibly switching fluorescent protein rsEGFP2 upon photoswitching is characterized by molecular modeling methods. Numerous conformations of the chromophore-binding site in computationally derived model systems are obtained using the quantum chemistry and QM/MM approaches. Excitation energies are computed using the extended multiconfigurational quasidegenerate perturbation theory (XMCQDPT2). The obtained structures and absorption spectra allow us to provide an interpretation of the observed structural and spectral properties of rsEGFP2 in the active ON and inactive OFF states. The results demonstrate that in addition to the dominating anionic and neutral forms of the chromophore, the cationic and zwitterionic forms may participate in the photoswitching of rsEGFP2. Conformations and protonation forms of the Glu223 and His149 side chains in the chromophore-binding site play an essential role in stabilizing specific protonation forms of the chromophore.

Photocycle in wild-type green fluorescent protein (wt-GFP) involves generation of a bright fluorescent deprotonated chromophore from feebly fluorescent protonated form via excited-state proton transfer. In addition to this usual photocycle, wt-GFP is also known to exhibit irreversible photoconversion upon illumination with ultraviolet and visible radiation. However, a detailed understanding of photoconversion in enhanced GFP (EGFP: S65T/F64L mutant of wt-GFP), which predominantly exists in deprotonated form, is yet to be explored. Using 254 nm irradiation, we study how photoconversion proceeds in EGFP. The key findings are observation of spreading out of an isosbestic point and existence of an initial lag phase in spectral kinetics of absorbance, indicative of sequential photoconversion through an intermediate. Fluorescence kinetics of EGFP and its photoproduct are estimated by assigning two unique fluorescence lifetimes which is further complicated by the fact that their fluorescence are spectrally inseparable, as evident from global analysis of fluorescence lifetime. Time-resolved fluorescence anisotropy studies further suggest minimal structural changes in protein scaffold upon photoconversion. Based on these findings, an analytic model is developed to account for the overall decay in fluorescence (as photoconversion proceeds) that inherently incorporates the initial lag phase and a summary of energetics and processes involved is provided., (© 2021 American Society for Photobiology.)

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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

Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO 2 , were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.

Green fluorescent protein (GFP) and its derivatives are widely used in biomedical research, and the manipulation of GFP-tagged proteins by GFP-specific binders is highly desired. However, structural information on how these binders bind with GFP is still lacking. In this study, we determined the crystal structure of the nanobody Nb2 complexed with superfolder GFP (sfGFP) at a resolution of 2.2 Å. Interestingly, although the complementarity-determining regions (CDRs) of Nb2 and LaG16 sequences were only 29.7% identical, they both bound to the same epitope of GFP and existed in the same orientation. Structural analysis indicated that they achieved similar binding characteristics through different mechanisms. We further verified the kinetics and thermodynamics of binding by biolayer interferometry (BLI) and isothermal titration calorimetry (ITC). Nb2 showed a slightly higher binding affinity for sfGFP than LaG16. The stability of GFP-specific nanobodies was verified by nano differential scanning fluorimetry (nanoDSF). Nb2 exhibited the highest melting temperature (Tm); thus, Nb2 is a promising GFP nanobody candidate for use in applications requiring harsh testing conditions. We also compared the binding sites of available GFP nanobodies and showed that some of them can simultaneously bind with GFP and assemble into multifunctional complexes to manipulate GFP-tagged target proteins. Our results provide atomic-scale binding information for Nb2-sfGFP, which is important for the further development of GFP-nanobody based fusion protein manipulation techniques., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2'-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO 2 Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO 2 Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO 2 Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63 + , CD81 + , or CD9 + EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50-170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Ratiometric genetically encoded calcium indicators (GECIs) record neural activity with high brightness while mitigating motion-induced artifacts. Recently developed ratiometric GECIs primarily employ cyan and yellow-fluorescent fluorescence resonance energy transfer pairs, and thus fall short in some applications that require deep tissue penetration and resistance to photobleaching. We engineered a set of green-red ratiometric calcium sensors that fused two fluorescent proteins and calcium sensing domain within an alternate configuration. The best performing elements of this palette of sensors, Twitch-GR and Twitch-NR, inherited the superior photophysical properties of their constituent fluorescent proteins. These properties enabled our sensors to outperform existing ratiometric calcium sensors in brightness and photobleaching metrics. In turn, the shot-noise limited signal fidelity of our sensors when reporting action potentials in cultured neurons and in the awake behaving mice was higher than the fidelity of existing sensors. Our sensor enabled a regime of imaging that simultaneously captured neural structure and function down to the deep layers of the mouse cortex., (© 2021. The Author(s).)

FGFRs are cell surface receptors that, when activated by specific FGFs ligands, transmit signals through the plasma membrane, regulating key cellular processes such as differentiation, division, motility, metabolism and death. We have recently shown that the modulation of the spatial distribution of FGFR1 at the cell surface constitutes an additional mechanism for fine-tuning cellular signaling. Depending on the multivalent, engineered ligand used, the clustering of FGFR1 into diverse supramolecular complexes enhances the efficiency and modifies the mechanism of receptor endocytosis, alters FGFR1 lifetime and modifies receptor signaling, ultimately determining cell fate. Here, we present a novel approach to generate multivalent FGFR1 ligands. We functionalized FGF1 for controlled oligomerization by developing N- and C-terminal fusions of FGF1 with the Fc fragment of human IgG1 (FGF1-Fc and Fc-FGF1). As oligomerization scaffolds, we employed GFPpolygons, engineered GFP variants capable of well-ordered multivalent display, fused to protein G to ensure binding of Fc fragment. The presented strategy allows efficient assembly of oligomeric FGFR1 ligands with up to twelve receptor binding sites. We show that multivalent FGFR1 ligands are biologically active and trigger receptor clustering on the cell surface. Importantly, the approach described in this study can be easily adapted to oligomerize alternative growth factors to control the activity of other cell surface receptors.

Since green fluorescent protein (GFP) has revolutionized molecular and cellular biology for about three decades, there has been a keen interest in understanding, designing, and controlling the fluorescence properties of GFP chromophore (i.e., HBDI) derivatives from the protein matrix to solution. Amongst these cross-disciplinary efforts, the elucidation of excited-state dynamics of HBDI derivatives holds the key to correlating the light-induced processes and fluorescence quantum yield (FQY). Herein, we implement steady-state electronic spectroscopy, femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and quantum calculations to study a series of mono- and dihalogenated HBDI derivatives (X = F, Cl, Br, 2F, 2Cl, and 2Br) in basic aqueous solution, gaining new insights into the photophysical reaction coordinates. In the excited state, the halogenated "floppy" chromophores exhibit an anti-heavy atom effect, reflected by strong correlations between FQY vs. Franck-Condon energy (EFC) or Stokes shift, and knrvs. EFC, as well as a swift bifurcation into the I-ring (major) and P-ring (minor) twisting motions. In the ground state, both ring-twisting motions become more susceptible to sterics and exhibit spectral signatures from the halogen-dependent hot ground-state absorption band decay in TA data. We envision this type of systematic analysis of the halogenated HBDI derivatives to provide guiding principles for the site-specific modification of GFP chromophores, and expand design space for brighter and potentially photoswitchable organic chemical probes in aqueous solution with discernible spectral signatures throughout the photocycle.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of l-lysine by l-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational l-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinked., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Japanese flounder are left-right asymmetrical, with features, such as dark, ocular-side specific pigmentation. This pigmentation arises during metamorphic stages, along with the asymmetric differentiation of adult-type chromatophores. Additionally, among juveniles, tank-reared specimens commonly show ectopic pigmentation on their blind sides. In both cases, neural crest-derived Sox10-positive progenitor cells at the dorsal fin base are hypothesized to contribute to chromatophore development. Here, we developed a method to visualize Sox10-positive cells via green fluorescent protein (GFP) fluorescence to directly monitor their migration and differentiation into chromatophores in vivo. Electroporation was applied to introduce GFP reporter vectors into the dorsal fin base of larvae and juveniles. Cre-loxP system vectors were also tested to enable cell labeling even after a decrease in sox10 expression levels. In larvae, undifferentiated Sox10-positive progenitor cells were labeled in the dorsal fin base, whereas newly differentiated adult-type chromatophores were seen dispersed on the ocular side. In juveniles, Sox10-positive cells were identified in the connective tissue of the dorsal fin base and observed prominently in areas of ectopic pigmentation, including several labeled melanophores. Thus, it was suggested that during metamorphic stages, Sox10-positive cells at the dorsal fin base contribute to adult-type chromatophore development, whereas in juveniles, they persist as precursors in the connective tissue, which in response to stimuli migrate to generate ectopic pigmentation. These findings contribute to elucidating pigmentation mechanisms, as well as abnormalities seen in hatchery-reared flounders. The electroporation method may be adapted to diverse animals as an accessible gene transfer method in various research fields, including developmental and biomedical studies., (© 2021 Wiley Periodicals LLC.)

The inhibition of the protein function for therapeutic applications remains challenging despite progress these past years. While the targeting application of molecularly imprinted polymer are in their infancy, no use was ever made of their magnetic hyperthermia properties to damage proteins when they are coupled to magnetic nanoparticles. Therefore, we have developed a facile and effective method to synthesize magnetic molecularly imprinted polymer nanoparticles using the green fluorescent protein (GFP) as the template, a bulk imprinting of proteins combined with a grafting approach onto maghemite nanoparticles. The hybrid material exhibits very high adsorption capacities and very strong affinity constants towards GFP. We show that the heat generated locally upon alternative magnetic field is responsible of the decrease of fluorescence intensity.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A 1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli , as well as for visualization of IBs interactions with eukaryotic cells.

Development of methodologies for optically triggered protein degradation enables the study of dynamic protein functions, such as those involved in cell signaling, that are difficult to be probed with traditional genetic techniques. Here, we describe the design and implementation of a novel light-controlled peptide degron conferring N-end pathway degradation to its protein target. The degron comprises a photocaged N-terminal amino acid and a lysine-rich, 13-residue linker. By caging the N-terminal residue, we were able to optically control N-degron recognition by an E3 ligase, consequently controlling ubiquitination and proteasomal degradation of the target protein. We demonstrate broad applicability by applying this approach to a diverse set of target proteins, including EGFP, firefly luciferase, the kinase MEK1, and the phosphatase DUSP6 (also known as MKP3). The caged degron can be used with minimal protein engineering and provides virtually complete, light-triggered protein degradation on a second to minute time scale.

The efficient and bioorthogonal chemical ligation reaction between potassium acyltrifluoroborates (KATs) and hydroxylamines (HAs) was used for the surface functionalization of a self-assembled monolayer (SAM) with biomolecules. An alkane thioether molecule with one terminal KAT group ( S-KAT ) was synthesized and adsorbed onto a gold surface, placing a KAT group on the top of the monolayer ( KAT-SAM ). As an initial test case, an aqueous solution of a hydroxylamine (HA) derivative of poly(ethylene glycol) (PEG) ( HA-PEG ) was added to this KAT-SAM at room temperature to perform the surface KAT ligation. Quartz crystal microbalance with dissipation (QCM-D) monitoring confirmed the rapid attachment of the PEG moiety onto the SAM. By surface characterization methods such as contact angle and ellipsometry, the attachment of PEG layer was confirmed, and covalent amide-bond formation was established by X-ray photoelectron spectroscopy (XPS). In a proof-of-concept study, the applicability of this surface KAT ligation for the attachment of biomolecules to surfaces was tested using a model protein, green fluorescent protein (GFP). A GFP was chemically modified with an HA linker to synthesize HA-GFP and added to the KAT-SAM under aqueous dilute conditions. A rapid attachment of the GFP on the surface was observed in real time by QCM-D. Despite the fact that such biomolecules have a variety of unprotected functional groups within their structures, the surface KAT ligation proceeded rapidly in a chemoselective manner. Our results demonstrate the versatility of the KAT ligation for the covalent attachment of a variety of water-soluble molecules onto SAM surfaces under dilute and biocompatible conditions to form stable, natural amide bonds.

Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021)., Competing Interests: S.J.B. is also scientific co-founder and VP Science Strategy at Artios Pharma Ltd., Babraham Research Campus, Cambridge, UK., (© 2021 The Authors.)

Cell differentiation, the process by which stem cells become specialized cells, is associated with chromatin reorganization inside the cell nucleus. Here, we measure the chromatin distribution and dynamics in embryonic stem cells in vivo before and after differentiation. We find that undifferentiated chromatin is less compact, more homogeneous, and more dynamic than differentiated chromatin. Furthermore, we present a noninvasive rheological analysis using intrinsic chromatin dynamics, which reveals that undifferentiated chromatin behaves like a Maxwell fluid, while differentiated chromatin shows a coexistence of fluidlike (sol) and solidlike (gel) phases. Our data suggest that chromatin undergoes a local sol-gel transition upon cell differentiation, corresponding to the formation of the more dense and transcriptionally inactive heterochromatin.

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

Green fluorescent protein (GFP) emits light when irradiated by not only light but also electrons. This electron-induced light emission called cathodoluminescence (CL) can be used to realize a high-resolution light emission microscopy based on the irradiation of a very narrow electron beam. To implement CL mapping in life sciences the investigation of the damage resistance of GFP to electron irradiation needs to be clarified. In this study, we investigated the electron radiation damage to GFP by analyzing the change in the CL intensity during electron beam irradiation. Since some of the CL spectra changed in shape during electron irradiation, the change in the intensity between 585 and 605 nm were measured. The characteristic doses at different electron current densities and electron energies were investigated. The characteristic dose of EGFP is much larger than that of coronene, which is one of the stable organic molecules against the electron beam irradiation., (Copyright © 2021. Published by Elsevier B.V.)

The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its p -HBI chromophore, corresponding to the cis and trans isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral trans chromophore at intermediate pH values, leading to ground-state cis-trans isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.

The gold nanoclusters (Au NCs) are a special kind of gold nanomaterial containing several gold atoms. Because of their small size and large surface area, Au NCs possess macroscopic quantum tunneling and dielectric domain effects. Furthermore, Au NCs fluorescent materials have longer luminous time and better photobleaching resistance compared with other fluorescent materials. The synthetic process of traditional Au NCs is complicated. Traditional Au NCs are prepared mainly by using polyamide amine type dendrites, and sixteen alkyl trimethylamine bromide or sulfhydryl small molecule as stabilizers. They are consequently synthesized by the reduction of strong reducing agents such as sodium borohydride. Notably, these materials are toxic and environmental-unfriendly. Therefore, there is an urgent need to develop more effective methods for synthesizing Au NCs via a green approach. On the other hand, the self-assembly of protein gold cluster-based materials, and their biomedical applications have become research hotspots in this field. We have been working on the synthesis, assembly and application of protein conjugated gold clusters for a long time. In this review, the synthesis and assembly of protein-gold nanoclusters and their usage in cell imaging and other medical research are discussed., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Extracellular vesicles (EVs) are valued candidates for the development of new tools for medical applications. Vesicles carrying melanoma-associated antigen A (MAGEA) proteins, a subfamily of cancer-testis antigens, are particularly promising tools in the fight against cancer. Here, we have studied the biophysical and chemical properties of MAGEA4-EVs and show that they are stable under common storage conditions such as keeping at +4 °C and -80 °C for at least 3 weeks after purification. The MAGEA4-EVs can be freeze-thawed two times without losing MAGEA4 in detectable quantities. The attachment of MAGEA4 to the surface of EVs cannot be disrupted by high salt concentrations or chelators, but the vesicles are sensitive to high pH. The MAGEA4 protein can bind to the surface of EVs in vitro, using robust passive incubation. In addition, EVs can be loaded with recombinant proteins fused to the MAGEA4 open reading frame within the cells and also in vitro. The high stability of MAGEA4-EVs ensures their potential for the development of EV-based anti-cancer applications.