Fluorescent protein (FP) variants have recently emerged as promising intracellular nitric oxide (NO) sensors based on NO-induced fluorescence loss due to cysteine S-nitrosylation - the covalent addition of a nitroso group to a cysteine thiol within a protein to form an S-nitrosothiol. Here, we investigate the mechanisms underlying this fluorescence loss using a combined experimental and computational approach. We focus on mTagBFP2, a blue fluorescent protein that undergoes a 70% reduction in fluorescence quantum yield and lifetime upon exposure to micromolar NO concentrations. We discriminate, through mutagenesis, the contributions of two key cysteine residues and propose an unprecedented excitation energy transfer (EET) from the mTagBFP2 chromophore to the S-nitroso groups as a potential quenching mechanism. Our EET efficiency calculations incorporate full couplings, the effects of the surrounding protein and solvent, and molecular dynamics-based configurational flexibility. The computed EET efficiencies broadly align with experimental observations, with remaining discrepancies for which we advance potential explanations. Our findings establish a mechanistic basis for NO-induced fluorescence loss in mTagBFP2, providing guidelines for the rational design of next-generation NO-sensitive FPs. Moreover, they suggest that analogous S-nitrosylation-driven quenching mechanisms could be operative in other FPs with exposed cysteine residues, underscoring the risk of artefacts in cellular imaging under physiological NO levels.

A missense variant in SLC1A3, which results in a proline to arginine substitution in the glial excitatory amino acid transporter 1 (EAAT1), causes a severe form of episodic ataxia type 6, characterized by recurrent attacks of ataxia and epilepsy. EAATs are dual function transport proteins, and the P290R variant reduces secondary active glutamate transport rates, while enhancing the anion channel activity. Here, we used complementary electrophysiological, imaging, biochemical and neuropathological techniques to characterize the cellular mechanisms underlying epileptic seizures in a mouse model of episodic ataxia type 6, the heterozygous Slc1a3P290R/+ mouse. Video-EEG recordings demonstrate frequent and severe spontaneous epileptic seizures in vivo. “Gliosis only” phenotype in the cerebrum of Slc1a3P290R/+ mice, which was restricted to the hippocampal formation, suggests that this brain structure may be involved in the development of epileptic seizure activity. Electrophysiological recordings from acute slices revealed a reduction in tonic GABAergic transmission in Slc1a3P290R/+ dentate gyrus granule cells, and to a lesser extent in cortical layer 2/3 pyramidal neurons before seizure onset. Phasic GABAergic and glutamatergic transmission remained unaltered in juvenile animals of the same developmental stage. There was no difference in expression levels of the GABAA receptor (GABAAR) δ subunits, suggesting that impaired tonic inhibition results from decreased extrasynaptic GABA concentrations. We identified enhanced GABA uptake by hippocampal radial glia-like cells (RGLs), caused by elevated GABA transporter 3 (GAT-3) expression and by an increased GABA transport driving force—due to lower intracellular chloride levels [Cl–]int – as cellular basis of impaired tonic inhibition. Our study demonstrates how increased EAAT1 chloride channel activity of the P290R variant can cause hyperexcitability by modifying synaptic GABA concentrations, while impaired EAAT1 glutamate transport leaves glutamatergic synaptic transmission in Slc1a3P290R/+ mice unaffected.

Abstract Although genetically encoded fluorescent sensors are fundamental tools to gain an understanding of intracellular nitric oxide (NO) dynamics, their development is still limited. We recently introduced the blue-emitting fluorescent protein (FP) mTagBFP2 as a NO sensor, where the chromophoric group formed upon S-nitrosylation of Cys residues leads to fluorescence quenching through excitation energy transfer. In this work, we further elaborated on this concept and devised a simple, two-domain, ratiometric NO sensor. mTagBFP2 was fused, with a suitable linker, to mCherry, a red-emitting fluorescent protein. As mCherry lacks cysteine residues, it is insensitive to NO and provides a convenient reference signal to normalize the response of mTagBFP2 fluorescence emission in a ratiometric detection. We demonstrate the capability of the construct to sense NO in the micromolar range in solution (Kd ca. 1 μM) and within mammalian (HeLa) and bacterial (Escherichia coli (E. coli)) cells.

Abstract The functional complexity of G protein-coupled receptors (GPCRs) arises from their structural dynamics, spanning timescales from nanoseconds to minutes. Single-molecule Förster Resonance Energy Transfer (smFRET) enables direct observation of these dynamics in individual receptors, either freely diffusing in solution, using confocal microscopy, or immobilized on surfaces, using Total Internal Reflection Fluorescence (TIRF) camera-based microscopy. However, these modalities are limited to distinct timescales – faster than milliseconds or slower than hundreds of milliseconds, respectively. To overcome these limitations, we employed smFRET with Anti-Brownian Electrokinetic (ABEL) trapping to extend the observation time of untethered human A2A adenosine receptors (A2AAR) reconstituted in lipid nanodiscs from milliseconds to seconds. We characterized conformational heterogeneity in apo and ligand-bound A2AAR and updated previous estimates of dwell times for long-lived receptor states from milliseconds to hundreds of milliseconds. Our results highlight the power of ABEL-FRET to probe GPCRs dynamics and offer valuable insights into GPCR conformational landscapes.

In this work, we combined plasmon-enhanced fluorescence and electrochemical (PEF-EC) trans- duction mechanisms to realize a highly sensitive dual-transducer aptasensor. To implement two traducers in one biosensor, a novel large-scale nanoimprint lithography process was introduced to fabricate gold nanopit arrays (AuNpA) with unique fringe structures. Light transmitting through the AuNpA samples exhibited a surface plasmon polariton peak overlapping with the excitation peak of the C7 aptamer associated fluorophore methylene blue (MB). We observed a five and seven times higher average fluorescence intensity over the AuNpA and fringe structure, respectively, in comparison to a plane Au film. Furthermore, the MB fluorophore was simultaneously utilized as a redox probe for electrochemical investigations and is described here as a dual transduction label for the first time. The novel dual transducer system was deployed for the detection of SARS-CoV-2 Spike protein via a C7 aptamer in combination with a strand displacement protocol. The PEF transducer exhibited a detection range from 1 fg/mL to 10 ng/mL with a detection limit of 0.07 fg/mL, while the EC traducer showed an extended dynamic range from 1 fg/mL to 100 ng/mL with a detection limit of 0.15 fg/mL. This work provides insights into an easy-to-perform, large-scale fabrication process for nanostructures enabling plasmon- enhanced fluorescence, and the development of an advanced but universal aptasensor platform.

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm 2 . Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

The primary role of telomerase is the lengthening of telomeres. Nonetheless, emerging evidence highlights additional functions of telomerase outside of the nucleus. Specifically, its catalytic subunit, TERT (Telomerase Reverse Transcriptase), is detected in the cytosol and mitochondria. Several studies have suggested an elevation in TERT concentration within mitochondria in response to oxidative stress. However, the origin of this mitochondrial TERT, whether transported from the nucleus or synthesized de novo, remains uncertain. In this study, we investigate the redistribution of TERT, labeled with a SNAP-tag, in response to oxidative stress using laser scanning fluorescence microscopy. Our findings reveal that, under our experimental conditions, there is no discernible transport of TERT from the nucleus to the mitochondria due to oxidative stress.

G-protein-coupled receptors (GPCRs) exhibit a variety of multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) can be applied to quantify the dynamics of individual receptor molecules in the presence or absence of different ligands. However, observation times of GPCRs, which are freely diffusing in solution, are limited to a few milliseconds. Here, we performed smFRET experiments on an active human A2A adenosine receptor reconstituted into a lipid nanodisc. We captured a single receptor with a confocal anti-Brownian electrokinetic trap (ABEL trap) and recorded smFRET time traces of up to seconds for individual receptors. Pulsed laser excitation enabled FRET donor lifetime recordings in parallel to confirm that conformational dynamics of the A2A adenosine receptor were causing smFRET fluctations. Influences of agonists as well as antagonists on transient conformational changes could be discriminated.

GABARAP, like other ATG8 proteins, is a ubiquitin-like modifier and its C-terminal lipid conjugation enables association with cellular membranes. To prevent interference with the lipidation process, N-terminal fluorescent protein (FP) tagging strategies have become the standard for studying ATG8 localization and function in living cells, significantly contributing to our understanding of this protein family's multifaceted roles. We employed live cell imaging with particular emphasis on a GABARAP split-tandem construct, GABARAP(G116A)-mTagBFP2-GABARAP (G-B-G), which retains both a free N-terminus and a lipidation-competent c-terminus, while bivalence creates a gain in affinity conferred by avidity. Notably, reminiscent of early in vitro studies demonstrating an interaction of GABARAP and tubulin, our results revealed a robust association of G-B-G with the microtubule network in living cells. We show that the presence of several basic residues in the amino-terminal helical subdomain of GABARAP and avidity emerged as essential for robust MT association, whereas lipidation ability was not decisive. Interestingly, while the position of the FP-tag had little influence on the result, the nature of the FP itself was crucial, with mTagBFP2 being required for tracking GABARAP tandems in the vicinity of MTs. Though artificial effects cannot be excluded, we assume that G-B-G, with its increased avidity, can give visibility to processes that are based on inherently weak interactions, and thus can help elucidate potential roles of GABARAP e.g. in microtubule-associated processes that are integral to autophagy-related and -unrelated cellular transport.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9′-norharmane dimer (nHo-dimer), which belongs to the β-carboline (βC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

The co-assembly of polyelectrolytes (PE) with proteins offers a promising approach for designing complex structures with customizable morphologies, charge distribution, and stability for targeted cargo delivery. However, the complexity of protein structure limits our ability to predict the properties of the formed nanoparticles, and our goal is to identify the key triggers of the morphological transition in protein/PE complexes and evaluate their ability to encapsulate multivalent ionic drugs. A positively charged PE can assemble with a protein at pH above isoelectric point due to the electrostatic attraction and disassemble at pH below isoelectric point due to the repulsion. The additional hydrophilic block of the polymer should stabilize the particles in solution and enable them to encapsulate a negatively charged drug in the presence of PE excess. We demonstrated that diblock copolymers, poly(ethylene oxide)block -poly(N,N-dimethylaminoethyl methacrylate) and poly(ethylene oxide)block -poly(N,N,N-trimethylammonioethyl methacrylate), consisting of a polycation block and a neutral hydrophilic block, reversibly co -assemble with insulin in pH range between 5 and 8. Using small -angle neutron and Xray scattering (SANS, SAXS), we showed that insulin arrangement within formed particles is controlled by intermolecular electrostatic forces between protein molecules, and can be tuned by varying ionic strength. For the first time, we observed by fluorescence that formed protein/PE complexes with excess of positive charges exhibited potential for encapsulating and controlled release of negatively charged bivalent drugs, protoporphyrin-IX and zinc(II) protoporphyrin-IX, enabling the development of nanocarriers for combination therapies with adjustable charge, stability, internal structure, and size.

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. Here we used thiol-reactive solvatochromic analogs of the green fluorescent protein (GFP) chromophore to track conformational changes in two proteins, recoverin and the A2A adenosine receptor (A2AAR). Two dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Our best-performing dye, DyeC, exhibited agonist-induced changes in both intensity and shape of its fluorescence spectrum when attached to A2AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the A2AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of A2AAR induced distinct fluorescence changes in the DyeC spectrum, indicating a unique receptor conformation. Our study demonstrated that GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), offering advantages such as intensity-based and ratiometric tracking, redshifted fluorescence spectra, and sensitivity to allosteric modulation.

GABARAP, like other ATG8 proteins, is a ubiquitin-like modifier and its C-terminal lipid conjugation enables association with cellular membranes. To prevent interference with the lipidation process, N-terminal fluorescent protein (FP) tagging strategies have become the standard for studying ATG8 localization and function in living cells, significantly contributing to our understanding of this protein family’s multifaceted roles. However, recent findings have unveiled potential limitations of bulky N-terminal tags, particularly regarding ATG8 functionality and localization in specific contexts. This study employed live cell imaging with particular emphasis on the GABARAP split-tandem construct, GABARAP(G116A)-mTagBFP2-GABARAP (G- B -G), which retains both a free N-terminus and a lipidation-competent C-terminus. Notably, our results revealed a robust association of G- B -G with the microtubule network in living cells which was not observed with N-terminal FP fusions of GABARAP, although early in vitro studies demonstrated an interaction of GABARAP and tubulin. Since we observed alteration of the microtubule network organization for G- B -G, this construct emerges as a valuable tool, which can help shedding light on potential roles of GABARAP in microtubule-associated processes that are integral to autophagy-related and -unrelated cellular transport.

G protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins in the human genome, and represent one of the most important classes of drug targets. Their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Therefore, in this study, we explored the efficacy of the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We selected the human A2A adenosine receptor (A2AAR), as a model protein, expressed it in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression. Overall, our results suggest that LEXSY is a promising platform for large-scale production of GPCRs for structural studies.

Extracellular potassium [K+]o elevation during synaptic activity retrogradely modifies presynaptic release and astrocytic uptake of glutamate. Hence, local K+ clearance and replenishment mechanisms are crucial regulators of glutamatergic transmission and plasticity. Based on recordings of astrocytic inward rectifier potassium current IKir and K+-sensitive electrodes as sensors of [K+]o as well as on in silico modeling, we demonstrate that the neuronal K+-Cl- co-transporter KCC2 clears local perisynaptic [K+]o during synaptic excitation by operating in an activity-dependent reversed mode. In reverse mode, KCC2 replenishes K+ in dendritic spines and complements clearance of [K+]o, therewith attenuating presynaptic glutamate release and shortening LTP. We thus demonstrate a physiological role of KCC2 in neuron-glial interactions and regulation of synaptic signaling and plasticity through the uptake of postsynaptically released K+.

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor's constitutive activity. For the agonist-bound A2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Abstract Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h−1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

Flavin-based fluorescent proteins (FbFPs), a class of small fluorescent proteins derived from light-oxygen-voltage (LOV) domains, bind ubiquitous endogenous flavins as chromophores. Due to their unique properties, they can be used as versatile in vivo reporter proteins under aerobic and anaerobic conditions. This chapter presents methodologies for in-depth characterization of the biochemical, spectroscopic, photophysical, and photochemical properties of FbFPs.