scholarly journals Dynamics of Germinosome Formation and FRET-Based Analysis of Interactions between GerD and Germinant Receptor Subunits in Bacillus cereus Spores

2021 ◽  
Vol 22 (20) ◽  
pp. 11230
Author(s):  
Yan Wang ◽  
Ronald M. P. Breedijk ◽  
Mark A. Hink ◽  
Lars Bults ◽  
Norbert O. E. Vischer ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Raw Materials ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescent Reporter ◽  
B Subunit ◽  
C Subunit ◽  
Bacterium Bacillus ◽  
And Function

Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores’ extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor’s (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)–SGFP2 and GerD–mScarlet-I, but there was no FRET between GerD–mScarlet-I and either GerRA–SGFP2 or GerRC–SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)–SGFP2 and GerD–mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)–SGFP2 and GerD–mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.

Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

2020 ◽  
Author(s):  
Lucas S. Ryan ◽  
Jeni Gerberich ◽  
Uroob Haris ◽  
ralph mason ◽  
Alexander Lippert
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Whole Body ◽  
Photon Flux ◽  
Ph Homeostasis ◽  
Ph Imaging ◽  
Confounding Variables ◽  
Significant Difference

<p>Regulation of physiological pH is integral for proper whole-body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, comprised of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8-8.4, making it viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished both using common fluorimetry and advanced optical imaging methods. Using an IVIS Spectrum, pH can be quantified through tissue with Ratio-pHCL-1, which has been shown in vitro and precisely calibrated in sacrificed mouse models. Initial studies showed that intraperitoneal injections of Ratio-pHCL-1 into sacrificed mice produce a photon flux of more than 10^10 photons per second, and showed a significant difference in ratio of emission intensities between pH 6.0, 7.0, and 8.0.</p> <b></b><i></i><u></u><sub></sub><sup></sup><br>

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

scholarly journals Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

scholarly journals Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Katie D. Hickey ◽  
Mary M. Buhr
Keyword(s):  
Lipid Bilayer ◽  
Lipid Composition ◽  
Protein Function ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Outer Leaflet ◽  
Lipid Environment ◽  
Protein Incorporation ◽  
And Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+K+-ATPase or -amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+K+-ATPase within the membranes.

scholarly journals Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

scholarly journals Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

2021 ◽  
Author(s):  
Agustina P. Bertolin ◽  
Florian Weissmann ◽  
Jingkun Zeng ◽  
Viktor Posse ◽  
Jennifer C. Milligan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Virus ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Etiologic Agent ◽  
Host Cells ◽  
Rdrp Activity ◽  
Chemical Library ◽  
Rna Dependent Rna Polymerase

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

scholarly journals Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure

2011 ◽  
Vol 195 (6) ◽  
pp. 1047-1060 ◽  
Author(s):  
Yoshifumi Yamaguchi ◽  
Naomi Shinotsuka ◽  
Keiko Nonomura ◽  
Kiwamu Takemoto ◽  
Keisuke Kuida ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Neural Tube ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Neural Tube Closure ◽  
Caspase Activation ◽  
Fluorescent Reporter ◽  
Time Lapse Imaging ◽  
Tube Closure

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)–based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.

scholarly journals Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Mitsutaka Kitano ◽  
Myra Hosmillo ◽  
Edward Emmott ◽  
Jia Lu ◽  
Ian Goodfellow
Keyword(s):  
Reverse Genetics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Murine Norovirus ◽  
Norwalk Virus ◽  
Irreversible Inhibitor ◽  
Antiviral Compounds ◽  
A Cell ◽  
First Time

ABSTRACT Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro . Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur.

scholarly journals Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

2018 ◽  
Vol 115 (40) ◽  
pp. 9923-9928 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Manu Ben Johny ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Single Type ◽  
Regulatory Subunits ◽  
And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

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