Prediction and characterization of the stability enhancing effect of the Cherry-Tag™ in highly concentrated protein solutions by complex rheological measurements and MD simulations

The conformational state of the activation loop (A-loop) is pivotal for the activity of most protein kinases. Hence, the characterization of the conformational dynamics of the A-loop is important to increase our understanding of the molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. Here, we carry out a combination of molecular dynamics (MD) and essential dynamics (ED) analyses to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the A-loop of Aurora-A. MD revealed that the stability of the A-loop in an open conformation is enhanced by single phospho-Thr-288, while paradoxically, the presence of a second phosphorylation at Thr-287 decreases such stability and renders the A-loop more fluctuant in time and space. Moreover, we found that this post-translational modification has a significant effect on the direction of the A-loop motions. ED analysis suggests that the presence of the phosphate moiety induces the dynamics of Aurora-A to sample two distinct energy minima, instead of a single large minimum, as in unphosphorylated Aurora-A states. This observation indicates that the conformational distributions of Aurora-A with both single and double phospho-threonine modifications are remarkably different from the unphosphorylated state. In the closed states, binding of CD532 and MLN8054 inhibitors has the effect of increasing the distance of the N- and C-lobes of the kinase domain of Aurora-A, and the angle analysis between those two lobes during MD simulations showed that the N- and C-lobes are kept more open in presence of CD532, compared to MLN8054. As the A-loop is a common feature of Aurora protein kinases, our studies provide a general description of the conformational dynamics of this structure upon phosphorylation and different ligands binding.

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Characterization of frozen hydrated biological specimens by low-loss EELS

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100132492 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1572-1573

Author(s):

Songquan Sun ◽

Richard D. Leapman

Keyword(s):

Water Content ◽

Direct Method ◽

Internal Standard ◽

Dry Weight ◽

Dark Field ◽

Protein Solutions ◽

Subcellular Compartment ◽

Differential Shrinkage ◽

Electron Energy Loss

Analyses of ultrathin cryosections are generally performed after freeze-drying because the presence of water renders the specimens highly susceptible to radiation damage. The water content of a subcellular compartment is an important quantity that must be known, for example, to convert the dry weight concentrations of ions to the physiologically more relevant molar concentrations. Water content can be determined indirectly from dark-field mass measurements provided that there is no differential shrinkage between compartments and that there exists a suitable internal standard. The potential advantage of a more direct method for measuring water has led us to explore the use of electron energy loss spectroscopy (EELS) for characterizing biological specimens in their frozen hydrated state.We have obtained preliminary EELS measurements from pure amorphous ice and from cryosectioned frozen protein solutions. The specimens were cryotransfered into a VG-HB501 field-emission STEM equipped with a 666 Gatan parallel-detection spectrometer and analyzed at approximately −160 C.

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TEM characterization of Au/Polysilicon interactions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100176381 ◽

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.

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Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM

10.26686/wgtn.12585593.v1 ◽

2020 ◽

Author(s):

MAK Williams ◽

V Cornuault ◽

AH Irani ◽

VV Symonds ◽

J Malmström ◽

...

Keyword(s):

Average Length ◽

American Chemical Society ◽

Md Simulations ◽

Chemical Society ◽

Side Chains ◽

Rhamnogalacturonan I ◽

Afm Imaging ◽

Minor Population ◽

The Stability ◽

A Minor

© 2020 American Chemical Society. Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 μm long was observed.

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

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191202145351 ◽

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.

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In Silico and in Vitro Evaluation of Deamidation Effects on the Stability of the Fusion Toxin DAB389IL-2

Current Proteomics ◽

10.2174/1570164616666190131150033 ◽

2019 ◽

Vol 16 (4) ◽

pp. 307-313 ◽

Cited By ~ 2

Author(s):

Nasrin Zarkar ◽

Mohammad Ali Nasiri Khalili ◽

Fathollah Ahmadpour ◽

Sirus Khodadadi ◽

Mehdi Zeinoddini

Keyword(s):

In Silico ◽

Catalytic Domain ◽

Md Simulations ◽

Special Importance ◽

Native Form ◽

Vitro Experiment ◽

In Vitro Experiment ◽

Pharmaceutical Protein ◽

The Stability

Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation. Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated. Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment. Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.

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Crystalline shielding mitigates structural rearrangement and localizes memory in jammed systems under oscillatory shear

Science Advances ◽

10.1126/sciadv.abe3392 ◽

2021 ◽

Vol 7 (20) ◽

pp. eabe3392

Author(s):

Erin G. Teich ◽

K. Lawrence Galloway ◽

Paulo E. Arratia ◽

Danielle S. Bassett

Keyword(s):

Local Structure ◽

Disordered Systems ◽

Amorphous Materials ◽

Structural Rearrangement ◽

Oscillatory Shear ◽

Disordered Materials ◽

Rheological Measurements ◽

Particle Level ◽

Individual Particles

The nature of yield in amorphous materials under stress has yet to be fully elucidated. In particular, understanding how microscopic rearrangement gives rise to macroscopic structural and rheological signatures in disordered systems is vital for the prediction and characterization of yield and the study of how memory is stored in disordered materials. Here, we investigate the evolution of local structural homogeneity on an individual particle level in amorphous jammed two-dimensional (athermal) systems under oscillatory shear and relate this evolution to rearrangement, memory, and macroscale rheological measurements. We define the structural metric crystalline shielding, and show that it is predictive of rearrangement propensity and structural volatility of individual particles under shear. We use this metric to identify localized regions of the system in which the material’s memory of its preparation is preserved. Our results contribute to a growing understanding of how local structure relates to dynamic response and memory in disordered systems.

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

Canadian Journal of Microbiology ◽

10.1139/m89-160 ◽

1989 ◽

Vol 35 (10) ◽

pp. 972-974 ◽

Cited By ~ 40

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.

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Characterization of Cocycle Attractors for Nonautonomous Reaction–Diffusion Equations

International Journal of Bifurcation and Chaos ◽

10.1142/s0218127416501352 ◽

2016 ◽

Vol 26 (08) ◽

pp. 1650135 ◽

Cited By ~ 2

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.

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Stability of Nonlinear Autonomous Quadratic Discrete Systems in the Critical Case

Discrete Dynamics in Nature and Society ◽

10.1155/2010/539087 ◽

2010 ◽

Vol 2010 ◽

pp. 1-23 ◽

Cited By ~ 11

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.

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