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 Intermolecular Interaction of Hthyni Protein with Double Methylated DNA at 5m-Cytosine Nucleotide

2020 ◽  
Vol 25 (1) ◽  
pp. 37-44
Author(s):  
Rajendra Prasad Koirala ◽  
Shyam Prakash Khanal ◽  
Sudip Shiwakoti ◽  
Narayan Prasad Adhikari
Keyword(s):  
Nuclear Protein ◽  
Hydrophobic Interactions ◽  
Md Simulations ◽  
Van Der Waals Interactions ◽  
Salt Bridges ◽  
Protein Residues ◽  
Methylated Dna ◽  
The Stability ◽  
Dna Complex ◽  
Binding Mechanisms

Human thymocyte nuclear protein 1 (hTHYN1) is one of the DNA binding proteins. It is essential for the regulation of Pax5 expression and the development of B cells in humans. Its thermodynamic and biological functions have been unclear yet. The study of the binding mechanism of hTHYN1 protein with DNA is essential to understand various biochemical functions in the human body. In this work, molecular dynamics (MD) simulations have been performed to understand the binding mechanisms of double methylated DNA (dmDNA) at cytosine nucleotide with hTHYN1 protein. Hydrogen bonding and other non-bonded (electrostatics and van der Waals) interactions among the residue-nucleotide pairs have been observed during the MD simulations and are also found responsible to form protein-DNA complex and to provide the stability of the structure. No salt bridges and hydrophobic interactions have been detected. Some of the protein residues in hTHYN1 have been found to strongly cooperate in the formation of the DNA-protein complex. Arginine residue of hTHYN1 has been observed as a major contributor in binding to the DNA. Many other residues also have significant roles in binding with DNA.

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.

scholarly journals Salt-Bridges in the Microenvironment of Stable Protein Structures

2020 ◽  
Vol 16 (11) ◽  
pp. 900-909
Author(s):  
Amal Kumar Bandyopadhyay ◽  
Keyword(s):  
Protein Structures ◽  
Salt Bridge ◽  
Homology Model ◽  
Net Energy ◽  
Salt Bridges ◽  
Site Specific ◽  
The Core ◽  
Polar Residues ◽  
The Stability

Salt-bridges (sb) play an important role in the folding and stability of proteins. This is deduced from the evaluation of net energy in the microenvironments (ME, residues that are 4Å away from positive and negative partners of salt-bridge and interact with them). ME’s act as a determinant of net-energy due to the intrinsic features by the sequence. The stability of extremophilic proteins is due to the presence of favorable residues at the ME without any unfavorable residues. We studied a dataset of four structures from the pdb and a homology model (PDB ID: 1HM5) to gain insights on this issue. Data shows that the presence of isolated charges and polar residues in the core of extremophilic proteins helps in the formation of stable salt-bridges with reduced desolvation. Thus, site-specific mutations with favorable residues at the ME will help develop thermo stable proteins with strong salt bridges.

TEM characterization of Au/Polysilicon interactions

1990 ◽  
Vol 48 (4) ◽  
pp. 650-651
Author(s):  
N. David Theodore ◽  
Leslie H. Allen ◽  
C. Barry Carter ◽  
James W. Mayer
Keyword(s):  
Solid Phase ◽  
Single Crystal Silicon ◽  
Chemical Vapor ◽  
Electrical Contacts ◽  
Silicon Substrates ◽  
Crystal Silicon ◽  
Transmission Electron ◽  
Polysilicon Films ◽  
The Stability

Metal/polysilicon investigations contribute to an understanding of issues relevant to the stability of electrical contacts in semiconductor devices. These investigations also contribute to an understanding of Si lateral solid-phase epitactic growth. Metals such as Au, Al and Ag form eutectics with Si. reactions in these metal/polysilicon systems lead to the formation of large-grain silicon. Of these systems, the Al/polysilicon system has been most extensively studied. In this study, the behavior upon thermal annealing of Au/polysilicon bilayers is investigated using cross-section transmission electron microscopy (XTEM). The unique feature of this system is that silicon grain-growth occurs at particularly low temperatures ∽300°C).Gold/polysilicon bilayers were fabricated on thermally oxidized single-crystal silicon substrates. Lowpressure chemical vapor deposition (LPCVD) at 620°C was used to obtain 100 to 400 nm polysilicon films. The surface of the polysilicon was cleaned with a buffered hydrofluoric acid solution. Gold was then thermally evaporated onto the samples.

Preparation and Characterization of β-glucosidase Films for Stabilization and Handling in Dry Configurations

2020 ◽  
Vol 21 (8) ◽  
pp. 741-747
Author(s):  
Liguang Zhang ◽  
Yanan Shen ◽  
Wenjing Lu ◽  
Lengqiu Guo ◽  
Min Xiang ◽  
...  
Keyword(s):  
Tensile Strength ◽  
Protein Function ◽  
Protein Stabilization ◽  
Functional Protein ◽  
Elongation At Break ◽  
Gelatin Films ◽  
The Stability ◽  
And Function ◽  
General Method

Background: Although the stability of proteins is of significance to maintain protein function for therapeutical applications, this remains a challenge. Herein, a general method of preserving protein stability and function was developed using gelatin films. Method: Enzymes immobilized onto films composed of gelatin and Ethylene Glycol (EG) were developed to study their ability to stabilize proteins. As a model functional protein, β-glucosidase was selected. The tensile properties, microstructure, and crystallization behavior of the gelatin films were assessed. Result: Our results indicated that film configurations can preserve the activity of β-glucosidase under rigorous conditions (75% relative humidity and 37°C for 47 days). In both control films and films containing 1.8 % β-glucosidase, tensile strength increased with increased EG content, whilst the elongation at break increased initially, then decreased over time. The presence of β-glucosidase had a negligible influence on tensile strength and elongation at break. Scanning electron-microscopy (SEM) revealed that with increasing EG content or decreasing enzyme concentrations, a denser microstructure was observed. Conclusion: In conclusion, the dry film is a promising candidate to maintain protein stabilization and handling. The configuration is convenient and cheap, and thus applicable to protein storage and transportation processes in the future.

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.

Effect of pH and temperature on the infectivity of human coronavirus 229E

1989 ◽  
Vol 35 (10) ◽  
pp. 972-974 ◽  
Author(s):  
Alain Lamarre ◽  
Pierre J. Talbot
Keyword(s):  
Incubation Period ◽  
Storage Conditions ◽  
Viral Infectivity ◽  
Human Coronavirus ◽  
Virus Growth ◽  
The Stability ◽  
And Storage ◽  
Influence Of Ph ◽  
Human Coronavirus 229E

The stability of human coronavirus 229E infectivity was maximum at pH 6.0 when incubated at either 4 or 33 °C. However, the influence of pH was more pronounced at 33 °C. Viral infectivity was completely lost after a 14-day incubation period at 22, 33, or 37 °C but remained relatively constant at 4 °C for the same length of time. Finally, the infectious titer did not show any significant reduction when subjected to 25 cycles of thawing and freezing. These studies will contribute to optimize virus growth and storage conditions, which will facilitate the molecular characterization of this important pathogen.Key words: coronavirus, pH, temperature, infectivity, human coronavirus.

scholarly journals Characterization of Cocycle Attractors for Nonautonomous Reaction–Diffusion Equations

2016 ◽  
Vol 26 (08) ◽  
pp. 1650135 ◽  
Author(s):  
C. A. Cardoso ◽  
J. A. Langa ◽  
R. Obaya
Keyword(s):  
Chaotic Dynamics ◽  
Linear Part ◽  
Reaction Diffusion ◽  
Positive Cone ◽  
Reaction Diffusion Equations ◽  
Diffusion Equations ◽  
Minimal Set ◽  
Full Characterization ◽  
The Stability

In this paper, we describe in detail the global and cocycle attractors related to nonautonomous scalar differential equations with diffusion. In particular, we investigate reaction–diffusion equations with almost-periodic coefficients. The associated semiflows are strongly monotone which allow us to give a full characterization of the cocycle attractor. We prove that, when the upper Lyapunov exponent associated to the linear part of the equations is positive, the flow is persistent in the positive cone, and we study the stability and the set of continuity points of the section of each minimal set in the global attractor for the skew product semiflow. We illustrate our result with some nontrivial examples showing the richness of the dynamics on this attractor, which in some situations shows internal chaotic dynamics in the Li–Yorke sense. We also include the sublinear and concave cases in order to go further in the characterization of the attractors, including, for instance, a nonautonomous version of the Chafee–Infante equation. In this last case we can show exponentially forward attraction to the cocycle (pullback) attractors in the positive cone of solutions.

scholarly journals Stability of Nonlinear Autonomous Quadratic Discrete Systems in the Critical Case

2010 ◽  
Vol 2010 ◽  
pp. 1-23 ◽  
Author(s):  
Josef Diblík ◽  
Denys Ya. Khusainov ◽  
Irina V. Grytsay ◽  
Zdenĕk Šmarda
Keyword(s):  
Lyapunov Functions ◽  
Critical Case ◽  
Autonomous Systems ◽  
Discrete Systems ◽  
Simple Eigenvalue ◽  
Discrete Equations ◽  
The Matrix ◽  
The Stability ◽  
Important Characterization

Many processes are mathematically simulated by systems of discrete equations with quadratic right-hand sides. Their stability is thought of as a very important characterization of the process. In this paper, the method of Lyapunov functions is used to derive classes of stable quadratic discrete autonomous systems in a critical case in the presence of a simple eigenvalueλ=1of the matrix of linear terms. In addition to the stability investigation, we also estimate stability domains.

Sign in / Sign up

Export Citation Format

Share Document