scholarly journals A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Markus Teucher ◽  
Hui Zhang ◽  
Verian Bader ◽  
Konstanze F. Winklhofer ◽  
Ana J. García-Sáez ◽  
...  
Keyword(s):  
Hela Cell ◽  
Quaternary Structure ◽  
Coarse Grained ◽  
Spin Labels ◽  
Cell Extracts ◽  
Protein Label ◽  
New Perspective ◽  
The Stability ◽  
Double Electron Electron Resonance

Abstract Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E. coli and HeLa cell extracts. We found that the gem-diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.

Aryl-Substituted and Benzo-Annulated CycloSal-Derivatives of 2′,3′-Dideoxy-2′,3′-Didehydrothymidine Monophosphate — Correlation of Structure, Hydrolysis Properties and Anti-HIV Activity

2002 ◽  
Vol 13 (2) ◽  
pp. 129-141 ◽  
Author(s):  
Christian Ducho ◽  
Jan Balzarini ◽  
Lieve Naesens ◽  
Erik De Clercq ◽  
Chris Meier
Keyword(s):  
Antiviral Activity ◽  
Degradation Products ◽  
Hydrolytic Stability ◽  
Substitution Pattern ◽  
Cell Extracts ◽  
Hydrolysis Process ◽  
The Stability ◽  
Anti Hiv

The synthesis of phenyl-substituted and benzo-annulated cycloSal phosphate triesters of the nucleoside analogue 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T, Zerit™) as lipophilic, membrane-soluble pronucleotides is described. The cycloSal moiety was introduced by using cyclic chlorophosphite agents prepared from phenyl-substituted saligenin derivatives and orthohydroxymethylated naphthols, respectively. Hydrolysis studies (HPLC analysis) of the triesters 2, 3 showed a range of hydrolytic stability from 1.4 h up to 5.1 h and the stability could be correlated with the substitution pattern in the cycloSal moiety. A slight decrease of their stability was observed, if phenyl-substituted derivatives were hydrolyzed in human CEM/O cell extracts. D4T and thymine, possible products of enzymatic cleavage of the pronucleotides, were not detected in the cell extracts. A further investigation of the hydrolysis process was performed by 31P-NMR spectroscopy. This technique allowed a precise monitoring of the degradation products and the exact determination of the product ratio. Finally, the newly synthesized compounds were tested concerning their antiviral activity against HIV in vitro. A strong correlation of the hydrolysis properties and the antiviral activity was found. 3-phenyl- cycloSal-d4TMP showed a threefold increase in its anti-HIV-1 activity and retained full activity in thymidine kinase (TK) deficient cells, indicative of a successful TK-bypass.

scholarly journals In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy

2020 ◽  
Vol 117 (34) ◽  
pp. 20566-20575 ◽  
Author(s):  
Yin Yang ◽  
Shen-Na Chen ◽  
Feng Yang ◽  
Xia-Yan Li ◽  
Akiva Feintuch ◽  
...  
Keyword(s):  
Protein Structures ◽  
Spin Labels ◽  
Salt Bridges ◽  
Protein Residues ◽  
Flexible Region ◽  
Charged Macromolecules ◽  
The Stability ◽  
Double Electron Electron Resonance ◽  
Bir Domain

The complexity of the cellular medium can affect proteins’ properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron–electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer–monomer dissociation constantKDwas markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.

scholarly journals In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

2020 ◽  
Author(s):  
Yin Yang ◽  
Shen-Na Chen ◽  
Feng Yang ◽  
Xia-Yan Li ◽  
Akiva Feintuch ◽  
...  
Keyword(s):  
Protein Structures ◽  
Salt Bridge ◽  
Spin Labels ◽  
Excluded Volume ◽  
Excluded Volume Effects ◽  
Distance Distributions ◽  
The Stability ◽  
Double Electron Electron Resonance ◽  
In Cells

AbstractThe complexity of the cellular medium can affect proteins’ properties and therefore in-cell characterization of proteins is essential. We explored the stability and conformation of BIR1, the first baculoviral IAP repeat domain of X-chromosome-linked inhibitor of apoptosis (XIAP), as a model for a homo-dimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three protein residues, C12 (flexible region), E22C and N28C (part of helical residues 26–31) in the N-terminal region. In contrast to predictions by excluded volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was recapitulated under conditions of high salt concentrations given that a conserved salt bridge at the dimer interface is critically required for association. Unexpectedly, however, also the addition of a crowding agent such as Ficoll destabilized the dimer, suggesting that Ficoll forms specific interactions with the monomeric protein. Changes in DEER distance distributions were observed for the E22C site, which displayed reduced conformational freedom in cells. Although overall DEER behaviors at E22C and N28C were compatible with a predicted compaction of disordered protein regions by excluded volume effects, we were unable to reproduce E22C properties in artificially crowded solutions. These results highlight the importance of in-cell DEER measurements to appreciate the complexities of cellular in vivo effects on protein structures and functions.

scholarly journals Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: A Molecular Simulation Study

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4299 ◽  
Author(s):  
Zeineb Si Chaib ◽  
Alessandro Marchetto ◽  
Klevia Dishnica ◽  
Paolo Carloni ◽  
Alejandro Giorgetti ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Transmembrane Protein ◽  
Translocator Protein ◽  
Coarse Grained ◽  
Mouse Protein ◽  
Two Factors ◽  
Domains Of Life ◽  
Contact Residues ◽  
The Stability

The translocator protein (TSPO) is a transmembrane protein present across the three domains of life. Its functional quaternary structure consists of one or more subunits. In mice, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

scholarly journals Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: a Molecular Simulation Study

2020 ◽  
Author(s):  
Zeineb Si Chaib ◽  
Alessandro Marchetto ◽  
Klevia Dishnica ◽  
Paolo Carloni ◽  
Alejandro Giorgetti ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Transmembrane Protein ◽  
Translocator Protein ◽  
Coarse Grained ◽  
Mouse Protein ◽  
Two Factors ◽  
Domains Of Life ◽  
Contact Residues ◽  
The Stability

The translocator protein (TSPO) is a transmembrane protein present in the three domains of life. Its functional quaternary structure consists of one or more subunits. In mouse, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

Determining the Norton’s Equivalent Model of Distribution System with Distributed Generation (DG) for Stability Analysis

2019 ◽  
Vol 12 (2) ◽  
pp. 190-198
Author(s):  
Sunny Katyara ◽  
Lukasz Staszewski ◽  
Faheem Akhtar Chachar
Keyword(s):  
Distributed Generation ◽  
Normal Condition ◽  
Distribution System ◽  
Distribution Networks ◽  
Equivalent Model ◽  
Stability Factor ◽  
Matlab Simulation ◽  
The Stability ◽  
Stability Issues

Background: Since the distribution networks are passive until Distributed Generation (DG) is not being installed into them, the stability issues occur in the distribution system after the integration of DG. Methods: In order to assure the simplicity during the calculations, many approximations have been proposed for finding the system’s parameters i.e. Voltage, active and reactive powers and load angle, more efficiently and accurately. This research presents an algorithm for finding the Norton’s equivalent model of distribution system with DG, considering from receiving end. Norton’s model of distribution system can be determined either from its complete configuration or through an algorithm using system’s voltage and current profiles. The algorithm involves the determination of derivative of apparent power against the current (dS/dIL) of the system. Results: This work also verifies the accuracy of proposed algorithm according to the relative variations in the phase angle of system’s impedance. This research also considers the varying states of distribution system due to switching in and out of DG and therefore Norton’s model needs to be updated accordingly. Conclusion: The efficacy of the proposed algorithm is verified through MATLAB simulation results under two scenarios, (i) normal condition and (ii) faulty condition. During normal condition, the stability factor near to 1 and change in dS/dIL was near to 0 while during fault condition, the stability factor was higher than 1 and the value of dS/dIL was away from 0.

Synthesis and solvatochromic studies of 2- amino-3-phenolazo1-(4-sulfophenyl)-3-methyi-5-pyrozolone and use it for the determination of trace amount of Nickel (II) in blood samples

2016 ◽  
Vol 5 (10) ◽  
pp. 4920
Author(s):  
Amar M. Ali ◽  
Hussain. J. Mohammed*
Keyword(s):  
Stability Constant ◽  
Trace Amount ◽  
Benzenesulfonic Acid ◽  
Determination Method ◽  
Blood Samples ◽  
Determination Of Nickel ◽  
Normal Human ◽  
The Stability ◽  
Normal Human Blood

A new, simple, sensitive and rapid spectrophotometric method is proposed for the determination of trace amount of Nickel (II). The method is based on the formation of a 1:2 complex with 4-(4-((2-hydroxy-6-nitrophenyl) diazenyl) -3-methyl-5-oxo-2, 5-dihydro-1H-pyrazol-1-yl) benzenesulfonic acid (2-ANASP) as a new reagent is developed. The complex has a maximum absorption at 516 nm and εmax of 1. 84 X 105 L. mol-1. cm-1. A linear correlation (0. 25 – 4. 0μg. ml-1) was found between absorbance at λmax and concentration. The accuracy and reproducibility of the determination method for various known amounts of Nickel (II) were tested. The results obtained are both precise (RSD was 1. 2 %) and accurate (relative error was 0. 787 %). The effect of diverse ions on the determination of Nickel (II) to investigate the selectivity of the method were also studied. The stability constant of the product was 0. 399 X 106 L. mol-1. The proposed method was successfully applied to the analysis of diabetes blood and normal human blood. 

scholarly journals DEER and RIDME Measurements of the Nitroxide-Spin Labelled Copper-Bound Amine Oxidase Homodimer from Arthrobacter Globiformis

2021 ◽  
Author(s):  
Hannah Russell ◽  
Rachel Stewart ◽  
Christopher Prior ◽  
Vasily S. Oganesyan ◽  
Thembaninkosi G. Gaule ◽  
...  
Keyword(s):  
Dipolar Coupling ◽  
Amine Oxidase ◽  
Spin Labels ◽  
Arthrobacter Globiformis ◽  
Copper Amine Oxidase ◽  
Dipole Interactions ◽  
Nitroxide Spin Labels ◽  
Double Electron Electron Resonance ◽  
Dipolar Modulation ◽  
Paramagnetic Centres

AbstractIn the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin–spin distances at nanometre-scale by measuring dipole–dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO.

scholarly journals Determination of Objective Weights Using a New Method Based on the Removal Effects of Criteria (MEREC)

Symmetry ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 525 ◽  
Author(s):  
Mehdi Keshavarz-Ghorabaee ◽  
Maghsoud Amiri ◽  
Edmundas Kazimieras Zavadskas ◽  
Zenonas Turskis ◽  
Jurgita Antucheviciene
Keyword(s):  
Essential Elements ◽  
New Method ◽  
Multi Criteria Decision Making ◽  
Analysis Of Means ◽  
Simulation Based ◽  
Criteria Weights ◽  
The Stability ◽  
Computational Analyses ◽  
Weighting Methods

The weights of criteria in multi-criteria decision-making (MCDM) problems are essential elements that can significantly affect the results. Accordingly, researchers developed and presented several methods to determine criteria weights. Weighting methods could be objective, subjective, and integrated. This study introduces a new method, called MEREC (MEthod based on the Removal Effects of Criteria), to determine criteria’ objective weights. This method uses a novel idea for weighting criteria. After systematically introducing the method, we present some computational analyses to confirm the efficiency of the MEREC. Firstly, an illustrative example demonstrates the procedure of the MEREC for calculation of the weights of criteria. Secondly, a comparative analysis is presented through an example for validation of the introduced method’s results. Additionally, we perform a simulation-based analysis to verify the reliability of MEREC and the stability of its results. The data of the MCDM problems generated for making this analysis follow a prevalent symmetric distribution (normal distribution). We compare the results of the MEREC with some other objective weighting methods in this analysis, and the analysis of means (ANOM) for variances shows the stability of its results. The conducted analyses demonstrate that the MEREC is efficient to determine objective weights of criteria.

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