Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An enzyme-based biosensor for monitoring and engineering protein stability in vivo.

ABSTRACT: Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysG^A) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.

SUBMITTER: Ren C

PROVIDER: S-EPMC8020752 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

In Vivo Detection of Glutamate in Tomatoes by an Enzyme-Based Electrochemical Biosensor.

Project description:The in vivo and on-site detection of key physiology parameters in plants will be of great relevance for precision agriculture and food technology. In this work, a sensitive enzymatic glutamate sensor was successfully developed. To enhance the conductivity and catalytic ability and to fix the glutamate oxidase, Au-Pt nanoparticles were first deposited on screen-printed electrodes, and then carboxylated graphene oxide and carboxylated multiwalled carbon nanotubes were fabricated for the synthesis of the electrode. The detection range of the glutamate sensor is widest (2 μM to 16 mM) up to date, and its detection limit is relatively low (0.14 μM). A number of standard curves were built in the pH range of 3.5-7.5, which can be applied in various plants and fruits. Using this sensor, the glutamate level in tomatoes was determined in vivo. This glutamate sensor has important practical value in precision agriculture. Our strategy also provides a way to establish the detection modes for other biomolecules in plants.

| S-EPMC9434751 | biostudies-literature

Protein Stability Effects in Aggregate-Based Enzyme Inhibition.

Project description:Small-molecule aggregates are a leading cause of artifacts in early drug discovery, but little is known about their interactions with proteins, nor why some proteins are more susceptible to inhibition than others. A possible reason for this apparent selectivity is that aggregation-based inhibition, as a stoichiometric process, is sensitive to protein concentration, which varies across assays. Alternatively, local protein unfolding by aggregates may lead to selectivity since stability varies among proteins. To deconvolute these effects, we used differentially stable point mutants of a single protein, TEM-1 ?-lactamase. Broadly, destabilized mutants had higher affinities for and were more potently inhibited by aggregates versus more stable variants. The addition of the irreversible inhibitor moxalactam destabilized several mutants, and these typically bound tighter to a colloidal particle, while the only mutant it stabilized bound weaker. These results suggest that less-stable enzymes are more easily sequestered and inhibited by colloidal aggregates.

| S-EPMC7156261 | biostudies-literature

Simultaneous in vivo enzyme and host engineering through laboratory evolution

Project description:Two genetic selection systems that couple metabolite hydroxylation or methylation of small molecules to growth of Escherichia coli are presented in this study. One system targets pterin-dependent hydroxylation (tBPt) while another focuses on S-adenosylmethionine-dependent methylation (SAM). Using adaptive laboratory evolution with growth selection, these two systems are demonstrated to not only achieve in vivo directed evolution of enzymes involved in human hormone biosynthesis but also reveal non-intuitive host factors that elude existing synthetic biology approaches. Raw sequencing data for the relevant strains generated in this study are presented here.

2019-01-30 | E-MTAB-6779 | biostudies-arrayexpress

Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.

Project description:In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FlAsH). Unfolding of labeled tetra-Cys CRABP I is accompanied by enhancement of FlAsH fluorescence, which made it possible to determine the free energy of unfolding of this protein by urea titration in cells and to follow in real time the formation of inclusion bodies by a slow-folding, aggregationprone mutant (FlAsH-labeled P39A tetra-Cys CRABP I). Aggregation in vivo displayed a concentration-dependent apparent lag time similar to observations of protein aggregation in purified in vitro model systems.

| S-EPMC327180 | biostudies-literature

Enzyme-Triggered Dissociation of a FRET-Based Protein Biosensor Monitored by Synchrotron SAXS.

Project description:Protein biosensors are widely used for the monitoring of metabolite concentration and enzymatic activities inside living cells and in in vitro applications. Neutrophil elastase (NE) is a serine protease of relevance in inflammatory diseases whose activity can lead to pathological conditions if unregulated. This study focuses on the structural characterization of a biosensor for NE activity based on Förster resonance energy transfer (FRET). The cleavage by NE results in dissociation of the FRET fluorescent protein pair and alteration of the fluorescent emission spectrum. We have used small angle x-ray scattering at a high intensity synchrotron source, combined with model-free analysis of the scattering data, to demonstrate the structure of the biosensor and the effect of its exposure to NE on size and shape. These investigations, together with biochemical studies, established the nanostructure-activity relationship that may contribute to the detailed understanding of the FRET-based biosensor and guide the rational design of new biosensor constructs.

| S-EPMC5647591 | biostudies-literature

Engineering an Enzyme for Direct Electrical Monitoring of Activity.

Project description:Proteins have been shown to be electrically conductive if tethered to an electrode by means of a specific binding agent, allowing single molecules to be wired into an electrical sensing circuit. Such circuits allow enzymes to be used as sensors, detectors, and sequencing devices. We have engineered contact points into a ?29 polymerase by introducing biotinylatable peptide sequences. The modified enzyme was bound to electrodes functionalized with streptavidin. ?29 connected by one biotinylated contact, and a second nonspecific contact showed rapid small fluctuations in current when activated. Signals were greatly enhanced with two specific contacts. Features in the distributions of DC conductance increased by a factor 2 or more over the open to closed conformational transition of the polymerase. Polymerase activity is manifested by a rapid (millisecond) large (25% of background) current fluctuations imposed on the DC conductance.

| S-EPMC7047563 | biostudies-literature

A SiO<sub>2</sub> Hybrid Enzyme-Based Biosensor with Enhanced Electrochemical Stability for Accuracy Detection of Glucose.

Project description:A novel enzyme-based biosensor for glucose detection is successfully developed using layer-by-layer assembly technology. The introduction of commercially available SiO2 was found to be a facile way to improve overall electrochemical stability. After 30 CV cycles, the proposed biosensor could retain 95% of its original current. The biosensor presents good detection stability and reproducibility with the detection concentration range of 1.96 × 10-9 to 7.24 × 10-7 M. This study demonstrated that the hybridization of cheap inorganic nanoparticles was a useful method in preparing high-performance biosensors with a much lower cost.

| S-EPMC10256442 | biostudies-literature

Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering.

Project description:In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic's Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.

| S-EPMC8553829 | biostudies-literature

A Superfolder Green Fluorescent Protein-Based Biosensor Allows Monitoring of Chloride in the Endoplasmic Reticulum.

Project description:Though the concentration of chloride has been measured in the cytoplasm and in secretory granules of live cells, it cannot be measured within the endoplasmic reticulum (ER) due to poor fluorescence of existing biosensors. We developed a fluorescent biosensor composed of a chloride-sensitive superfolder GFP and long Stokes-shifted mKate2 for simultaneous chloride and pH measurements that retained fluorescence in the ER lumen. Using this sensor, we showed that the chloride concentration in the ER is significantly lower than that in the cytosol. This improved biosensor enables dynamic measurement of chloride in the ER and may be useful in other environments where protein folding is challenging.

| S-EPMC9425558 | biostudies-literature

Novel yeast-based biosensor for environmental monitoring of tebuconazole.

Project description:Due to increasing demand for high and stable crop production, human populations are highly dependent on pesticide use for growing and storing food. Environmental monitoring of these agrochemicals is therefore of utmost importance, because of their collateral effects on ecosystem and human health. Even though most current-use analytical methods achieve low detection limits, they require procedures that are too complex and costly for routine monitoring. As such, there has been an increased interest in biosensors as alternative or complementary tools to streamline detection and quantification of environmental contaminants. In this work, we developed a biosensor for environmental monitoring of tebuconazole (TEB), a common agrochemical fungicide. For that purpose, we engineered S. cerevisiae cells with a reporter gene downstream of specific promoters that are expressed after exposure to TEB and characterized the sensitivity and specificity of this model system. After optimization, we found that this easy-to-use biosensor consistently detects TEB at concentrations above 5 μg L-1 and does not respond to realistic environmental concentrations of other tested azoles, suggesting it is specific. We propose the use of this system as a complementary tool in environmental monitoring programs, namely, in high throughput scenarios requiring screening of numerous samples. KEY POINTS: • A yeast-based biosensor was developed for environmental monitoring of tebuconazole. •The biosensor offers a rapid and easy method for tebuconazole detection ≥ 5 μg L-1. •The biosensor is specific to tebuconazole at environmentally relevant concentrations.

| S-EPMC10764535 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data