scholarly journals Making Soup: Preparing and Validating Molecular Simulations of the Bacterial Cytoplasm

2019 ◽  
Author(s):  
Leandro Oliveira Bortot ◽  
Zahedeh Bashardanesh ◽  
David van der Spoel
Keyword(s):  
Large Scale ◽  
Biological Properties ◽  
Computational Power ◽  
E Coli ◽  
Solvent Models ◽  
Explicit Solvent ◽  
Computational Modeling And Simulation ◽  
Dynamics Simulations ◽  
The Individual ◽  
Bacterial Cytoplasm

Biomolecular crowding affects the biophysical and biochemical behavior of macro- molecules when compared to the dilute environment present in experiments made with isolated proteins. Computational modeling and simulation are useful tools to study how crowding affects the structural dynamics and biological properties of macromolecules. As computational power increased, modeling and simulating large scale all-atom explicit solvent models of the prokaryote cytoplasm become possible. In this work, we build an atomistic model of the cytoplasm of Escherichia coli composed of 1.5 million atoms and submit it to a total of 3 μs of molecular dynamics simulations. The properties of biomolecules under crowding conditions are compared to those from simulations of the individual compounds under dilute conditions. The simulation model is found to be consistent with experimental data about the diffusion coefficient and stability of macromolecules under crowded conditions. In order to stimulate further work we provide a Python script and a set of files that enables other researchers to build their own E. coli cytoplasm models to address questions related to crowding.<br>

scholarly journals Making Soup: Preparing and Validating Molecular Simulations of the Bacterial Cytoplasm

2019 ◽  
Author(s):  
Leandro Oliveira Bortot ◽  
Zahedeh Bashardanesh ◽  
David van der Spoel
Keyword(s):  
Large Scale ◽  
Biological Properties ◽  
Computational Power ◽  
E Coli ◽  
Solvent Models ◽  
Explicit Solvent ◽  
Computational Modeling And Simulation ◽  
Dynamics Simulations ◽  
The Individual ◽  
Bacterial Cytoplasm

Biomolecular crowding affects the biophysical and biochemical behavior of macro- molecules when compared to the dilute environment present in experiments made with isolated proteins. Computational modeling and simulation are useful tools to study how crowding affects the structural dynamics and biological properties of macromolecules. As computational power increased, modeling and simulating large scale all-atom explicit solvent models of the prokaryote cytoplasm become possible. In this work, we build an atomistic model of the cytoplasm of Escherichia coli composed of 1.5 million atoms and submit it to a total of 3 μs of molecular dynamics simulations. The properties of biomolecules under crowding conditions are compared to those from simulations of the individual compounds under dilute conditions. The simulation model is found to be consistent with experimental data about the diffusion coefficient and stability of macromolecules under crowded conditions. In order to stimulate further work we provide a Python script and a set of files that enables other researchers to build their own E. coli cytoplasm models to address questions related to crowding.<br>

scholarly journals Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5538
Author(s):  
Zhongxuan Li ◽  
Qiang Cheng ◽  
Henan Guo ◽  
Rijun Zhang ◽  
Dayong Si
Keyword(s):  
Pichia Pastoris ◽  
High Performance ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
E Coli ◽  
Cell Membrane Permeabilization ◽  
Bactericidal Activities ◽  
The Individual ◽  
Industrial Level

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

scholarly journals The Role of Displacing Confined Solvent in the Conformational Equilibrium of β-Cyclodextrin

2019 ◽  
Author(s):  
Peng He ◽  
Sheila Sarkar ◽  
Emilio Gallicchio ◽  
Tom Kurtzman ◽  
Lauren Wickstrom
Keyword(s):  
Conformational Equilibrium ◽  
Implicit Solvent ◽  
Theory Analysis ◽  
State Behavior ◽  
Generalized Born ◽  
Solvent Models ◽  
Explicit Solvent ◽  
Dynamics Simulations ◽  
Opening And Closing

<p>This study investigates the role of hydration and its relationship to the conformational equilibrium of the host molecule β-cyclodextrin. Molecular dynamics simulations indicate that the unbound β-cyclodextrin exhibits two state behavior in explicit solvent due to the opening and closing of its cavity. In implicit solvent, these transitions are not observed and there is one dominant conformation of β-cyclodextrin with an open cavity. Based on these observations, we investigate the hypothesis that the expulsion of thermodynamically unfavorable water molecules into the bulk plays an important role in controlling the accessibility of the closed macrostate at room temperature. We compare the results of the molecular mechanics analytical generalized Born plus non-polar solvation approach to those obtained through Grid Inhomogeneous Solvation Theory analysis with explicit solvation to elucidate the thermodynamic forces at play. The calculations help to illustrate the deficiencies of continuum solvent models and demonstrate the key role of the thermodynamics of enclosed hydration in driving the conformational equilibrium of molecules in solution. </p>

scholarly journals His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?

2017 ◽  
Vol 2017 ◽  
pp. 1-12
Author(s):  
Elena Krachmarova ◽  
Milena Tileva ◽  
Elena Lilkova ◽  
Peicho Petkov ◽  
Klaus Maskos ◽  
...  
Keyword(s):  
Interferon Gamma ◽  
Biological Properties ◽  
Expression Vectors ◽  
Human Interferon ◽  
Fusion Partner ◽  
E Coli ◽  
Binding Domains ◽  
Close Proximity ◽  
Dynamics Simulations ◽  
Shed Light

In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed inE. colicells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγreceptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.

scholarly journals Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Hanne L. P. Tytgat ◽  
Chia-wei Lin ◽  
Mikail D. Levasseur ◽  
Markus B. Tomek ◽  
Christoph Rutschmann ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Biomedical Applications ◽  
Biological Properties ◽  
Cytoplasmic Localization ◽  
Periplasmic Space ◽  
Site Specific ◽  
E Coli ◽  
Self Assembling ◽  
Mammalian Origin ◽  
Bacterial Cytoplasm

AbstractGlycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

scholarly journals Conserved Dynamic Mechanism of Allosteric Response to L-arg in Divergent Bacterial Arginine Repressors

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2247
Author(s):  
Saurabh Kumar Pandey ◽  
Milan Melichercik ◽  
David Řeha ◽  
Rüdiger H. Ettrich ◽  
Jannette Carey
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Large Scale ◽  
Rotational Transition ◽  
Sequence Motifs ◽  
E Coli ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Distinct Sequence ◽  
Dynamics Simulations

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.

scholarly journals The Distal Polybasic Cleavage Sites of SARS-CoV-2 Spike Protein Enhance Spike Protein-ACE2 Binding

2020 ◽  
Author(s):  
Baofu Qiao ◽  
Monica Olvera de la Cruz
Keyword(s):  
Viral Entry ◽  
Electrostatic Interactions ◽  
Large Scale ◽  
Cell Receptor ◽  
Spike Protein ◽  
Cleavage Sites ◽  
Explicit Solvent ◽  
Dynamics Simulations ◽  
Positively Charged

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD-ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein-ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD-ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD-ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD-ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection.Abstract FigureTOC:The SARS-CoV-2 spike protein-ACE2 complex showing the polybasic cleavage sites

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