scholarly journals MreC and MreD balance the interaction between the elongasome proteins PBP2 and RodA

2019 ◽  
Author(s):  
Xiaolong Liu ◽  
Jacob Biboy ◽  
Waldemar Vollmer ◽  
Tanneke den Blaauwen
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Cell Envelope ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cylindrical Part ◽  
E Coli ◽  
Length Growth ◽  
Antibiotic Drug

AbstractRod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a chance in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the “on state” of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.ImportanceThe cell envelope of Escherichia coli bears the protective and shape-determining peptidoglycan layer sandwiched between the outer and inner membranes. Length growth in bacteria is accomplished by a protein complex termed elongasome. We used Förster Resonance Energy Transfer (FRET) that reports not only on whether proteins interact with each other but also on conformational changes during interactions, to investigate how the elongasome might be activated. RodA and PBP2 provide the peptidoglycan glycosyltransferase and transpeptidase activities needed to synthesize new peptidolgycan during length growth, respectively, and PBP2 activates RodA. We show that the interactions between MreC and MreD with PBP2-RodA alter the nature of the interaction between PBP2 and RodA and hypothesis that the corresponding conformational change in the PBP2-RodA complex allows switching between the ‘on’ and ‘off’ states of the elongasome.

scholarly journals Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells

2007 ◽  
Vol 176 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Dawen Cai ◽  
Adam D. Hoppe ◽  
Joel A. Swanson ◽  
Kristen J. Verhey
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Structural Organization ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Protein Activity ◽  
Kinesin Light Chain ◽  
Initial Interaction

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)–labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.

scholarly journals Biosensors-Based In Vivo Quantification of 2-Oxoglutarate in Cyanobacteria and Proteobacteria

Life ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 51 ◽  
Author(s):  
Hai-Lin Chen ◽  
Amel Latifi ◽  
Cheng-Cai Zhang ◽  
Christophe Bernard
Keyword(s):  
Conformational Changes ◽  
Nitrogen Assimilation ◽  
Krebs Cycle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Level ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

2-oxoglutarate (α-ketoglutarate; 2-OG) is an intermediate of the Krebs cycle, and constitutes the carbon skeleton for nitrogen assimilation and the synthesis of a variety of compounds. In addition to being an important metabolite, 2-OG is a signaling molecule with a broad regulatory repertoire in a variety of organisms, including plants, animals, and bacteria. Although challenging, measuring the levels and variations of metabolic signals in vivo is critical to better understand how cells control specific processes. To measure cellular 2-OG concentrations and dynamics, we designed a set of biosensors based on the fluorescence resonance energy transfer (FRET) technology that can be used in vivo in different organisms. For this purpose, we took advantage of the conformational changes of two cyanobacterial proteins induced by 2-OG binding. We show that these biosensors responded immediately and specifically to different 2-OG levels, and hence allowed to measure 2-OG variations in function of environmental modifications in the proteobacterium Escherichia coli and in the cyanobacterium Anabaena sp. PCC 7120. Our results pave the way to study 2-OG dynamics at the cellular level in uni- and multi-cellular organisms.

scholarly journals The integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Claudine Fraipont ◽  
Svetlana Alexeeva ◽  
Benoît Wolf ◽  
René van der Ploeg ◽  
Marie Schloesser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cylindrical Part ◽  
Class A ◽  
Lipid Ii ◽  
Class B

During the cell cycle of rod-shaped bacteria, two morphogenetic processes can be discriminated: length growth of the cylindrical part of the cell and cell division by formation of two new cell poles. The morphogenetic protein complex responsible for the septation during cell division (the divisome) includes class A and class B penicillin-binding proteins (PBPs). In Escherichia coli, the class B PBP3 is specific for septal peptidoglycan synthesis. It requires the putative lipid II flippase FtsW for its localization at the division site and is necessary for the midcell localization of the class A PBP1B. In this work we show direct interactions between FtsW and PBP3 in vivo and in vitro by FRET (Förster resonance energy transfer) and co-immunoprecipitation experiments. These proteins are able to form a discrete complex independently of the other cell-division proteins. The K2–V42 peptide of PBP3 containing the membrane-spanning sequence is a structural determinant sufficient for interaction with FtsW and for PBP3 dimerization. By using a two-hybrid assay, the class A PBP1B was shown to interact with FtsW. However, it could not be detected in the immunoprecipitated FtsW–PBP3 complex. The periplasmic loop 9/10 of FtsW appeared to be involved in the interaction with both PBP1B and PBP3. It might play an important role in the positioning of these proteins within the divisome.

scholarly journals Spatial and Temporal Regulation of Focal Adhesion Kinase Activity in Living Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 201-214 ◽  
Author(s):  
Xinming Cai ◽  
Daniel Lietha ◽  
Derek F. Ceccarelli ◽  
Andrei V. Karginov ◽  
Zenon Rajfur ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Conformational Change ◽  
Focal Adhesion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Live Cells ◽  
Major Mechanism

ABSTRACT Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.

scholarly journals Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum

Life ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 147 ◽  
Author(s):  
Fabiana Miraglia ◽  
Verdiana Valvano ◽  
Lucia Rota ◽  
Cristina Di Primio ◽  
Valentina Quercioli ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Stable Cell Line ◽  
Fret Biosensors ◽  
Alpha Synuclein Aggregation ◽  
Small Subpopulation

Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (αS) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of αS resulted in the formation of compact αS-positive structures in a small subpopulation of cells, resembling αS inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of αS FRET biosensors (AFBs) able to track αS conformation in cells. In native conditions, expression in i36, a stable cell line for ER αS, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of αS (αS-CFP/αS-YFP), resulted in no Förster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with αS N- or C- termini (YFP-αS-CFP), showed a positive FRET signal. These data confirmed that αS has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of αS oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in αS conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track αS conformational changes in vivo.

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 Multiple system atrophy-associated oligodendroglial protein p25α stimulates formation of novel α-synuclein strain with enhanced neurodegenerative potential

2021 ◽  
Author(s):  
Nelson Ferreira ◽  
Hjalte Gram ◽  
Zachary A. Sorrentino ◽  
Emil Gregersen ◽  
Sissel Ida Schmidt ◽  
...  
Keyword(s):  
Multiple System Atrophy ◽  
Protein Misfolding ◽  
Resonance Energy Transfer ◽  
Lewy Bodies ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Striatal Neurons ◽  
End Stage ◽  
Intracellular Aggregates

AbstractPathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a “tropism” for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

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 Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

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