ResearchPad - electrostatics https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[The role of the C<sub>2</sub>A domain of synaptotagmin 1 in asynchronous neurotransmitter release]]> https://www.researchpad.co/article/elastic_article_14623 Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.

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<![CDATA[PEO-b-PPO star-shaped polymers enhance the structural stability of electrostatically coupled liposome/polyelectrolyte complexes]]> https://www.researchpad.co/article/5c605a4ad5eed0c4847ccce0

We propose a strategy to counteract the salt-driven disassembly of multiliposomal complexes made by electrostatic co-assembly of anionic small unilamellar liposomes and cationic star-shaped polyelectrolytes (made of quaternized poly(dimethylaminoethyl methacrylate) (qPDMAEMA100)3.1). The combined action of (qPDMAEMA100)3.1 and a nonionic star-shaped polymer (PEO12-b-PPO45)4, which comprises diblock copolymer arms uniting a poly(ethylene oxide) PEO inner block and a poly(propylene oxide) PPO terminal block, leads to a stabilization of these complexes against disintegration in saline solutions. Hereby, the anchoring of the PPO terminal blocks to the lipid bilayer and the bridging between several liposomes are at the origin of the promoted structural stability. Two-focus fluorescence correlation spectroscopy verifies the formation of multiliposomal complexes with (PEO12-b-PPO45)4. The polyelectrolyte and the amphiphilic polymer work synergistically, as the joint action still assures some membrane integrity, which is not seen for the mere (PEO12-b-PPO45)4—liposome interaction alone.

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<![CDATA[Structural characterization of a pathogenicity-related superoxide dismutase codified by a probably essential gene in Xanthomonas citri subsp. citri]]> https://www.researchpad.co/article/5c3d015bd5eed0c48403a8e5

Citrus canker is a plant disease caused by the bacteria Xanthomonas citri subsp. citri that affects all domestic varieties of citrus. Some annotated genes from the X. citri subsp. citri genome are assigned to an interesting class named "pathogenicity, virulence and adaptation". Amongst these is sodM, which encodes for the gene product XcSOD, one of four superoxide dismutase homologs predicted from the genome. SODs are widespread enzymes that play roles in the oxidative stress response, catalyzing the degradation of the deleterious superoxide radical. In Xanthomonas, SOD has been associated with pathogenesis as a counter measure against the plant defense response. In this work we initially present the 1.8 Å crystal structure of XcSOD, a manganese containing superoxide dismutase from Xanthomonas citri subsp. citri. The structure bears all the hallmarks of a dimeric member of the MnSOD family, including the conserved hydrogen-bonding network residues. Despite the apparent gene redundancy, several attempts to obtain a sodM deletion mutant were unsuccessful, suggesting the encoded protein to be essential for bacterial survival. This intriguing observation led us to extend our structural studies to the remaining three SOD homologs, for which comparative models were built. The models imply that X. citri subsp. citri produces an iron-containing SOD which is unlikely to be catalytically active along with two conventional Cu,ZnSODs. Although the latter are expected to possess catalytic activity, we propose they may not be able to replace XcSOD for reasons such as distinct subcellular compartmentalization or differential gene expression in pathogenicity-inducing conditions.

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<![CDATA[Energy landscape for the insertion of amphiphilic nanoparticles into lipid membranes: A computational study]]> https://www.researchpad.co/article/5c3fa610d5eed0c484cabadc

Amphiphilic, monolayer-protected gold nanoparticles (NPs) have been shown to enter cells via a non-endocytic, non-disruptive pathway that could be valuable for biomedical applications. The same NPs were also found to insert into a series of model cell membranes as a precursor to cellular uptake, but the insertion mechanism remains unclear. Previous simulations have demonstrated that an amphiphilic NP can insert into a single leaflet of a planar lipid bilayer, but in this configuration all charged end groups are localized to one side of the bilayer and it is unknown if further insertion is thermodynamically favorable. Here, we use atomistic molecular dynamics simulations to show that an amphiphilic NP can reach the bilayer midplane non-disruptively if charged ligands iteratively “flip” across the bilayer. Ligand flipping is a favorable process that relaxes bilayer curvature, decreases the nonpolar solvent-accessible surface area of the NP monolayer, and increases attractive ligand-lipid electrostatic interactions. Analysis of end group hydration further indicates that iterative ligand flipping can occur on experimentally relevant timescales. Supported by these results, we present a complete energy landscape for the non-disruptive insertion of amphiphilic NPs into lipid bilayers. These findings will help guide the design of NPs to enhance bilayer insertion and non-endocytic cellular uptake, and also provide physical insight into a possible pathway for the translocation of charged biomacromolecules.

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<![CDATA[Amino acid residues in five separate HLA genes can explain most of the known associations between the MHC and primary biliary cholangitis]]> https://www.researchpad.co/article/5c0ed757d5eed0c484f13f5f

Primary Biliary Cholangitis (PBC) is a chronic autoimmune liver disease characterised by progressive destruction of intrahepatic bile ducts. The strongest genetic association is with HLA-DQA1*04:01, but at least three additional independent HLA haplotypes contribute to susceptibility. We used dense single nucleotide polymorphism (SNP) data in 2861 PBC cases and 8514 controls to impute classical HLA alleles and amino acid polymorphisms using state-of-the-art methodologies. We then demonstrated through stepwise regression that association in the HLA region can be largely explained by variation at five separate amino acid positions. Three-dimensional modelling of protein structures and calculation of electrostatic potentials for the implicated HLA alleles/amino acid substitutions demonstrated a correlation between the electrostatic potential of pocket P6 in HLA-DP molecules and the HLA-DPB1 alleles/amino acid substitutions conferring PBC susceptibility/protection, highlighting potential new avenues for future functional investigation.

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<![CDATA[Temperature Modulation of Electric Fields in Biological Matter]]> https://www.researchpad.co/article/5989dafeab0ee8fa60bc5bd2

Pulsed electric fields (PEF) have become an important minimally invasive surgical technology for various applications including genetic engineering, electrochemotherapy and tissue ablation. This study explores the hypothesis that temperature dependent electrical parameters of tissue can be used to modulate the outcome of PEF protocols, providing a new means for controlling and optimizing this minimally invasive surgical procedure. This study investigates two different applications of cooling temperatures applied during PEF. The first case utilizes an electrode which simultaneously delivers pulsed electric fields and cooling temperatures. The subsequent results demonstrate that changes in electrical properties due to temperature produced by this configuration can substantially magnify and confine the electric fields in the cooled regions while almost eliminating electric fields in surrounding regions. This method can be used to increase precision in the PEF procedure, and eliminate muscle contractions and damage to adjacent tissues. The second configuration considered introduces a third probe that is not electrically active and only applies cooling boundary conditions. This second study demonstrates that in this probe configuration the temperature induced changes in electrical properties of tissue substantially reduce the electric fields in the cooled regions. This novel treatment can potentially be used to protect sensitive tissues from the effect of the PEF. Perhaps the most important conclusion of this investigation is that temperature is a powerful and accessible mechanism to modulate and control electric fields in biological tissues and can therefore be used to optimize and control PEF treatments.

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<![CDATA[Evidences of Changes in Surface Electrostatic Charge Distribution during Stabilization of HPV16 Virus-Like Particles]]> https://www.researchpad.co/article/5989d9ddab0ee8fa60b685e9

The stabilization of human papillomavirus type 16 virus-like particles has been examined by means of different techniques including dynamic and static light scattering, transmission electron microscopy and electrophoretic mobility. All these techniques provide different and often complementary perspectives about the aggregation process and generation of stabilized virus-like particles after a period of time of 48 hours at a temperature of 298 K. Interestingly, static light scattering results point towards a clear colloidal instability in the initial systems, as suggested by a negative value of the second virial coefficient. This is likely related to small repulsive electrostatic interactions among the particles, and in agreement with relatively small absolute values of the electrophoretic mobility and, hence, of the net surface charges. At this initial stage the small repulsive interactions are not able to compensate binding interactions, which tend to aggregate the particles. As time proceeds, an increase of the size of the particles is accompanied by strong increases, in absolute values, of the electrophoretic mobility and net surface charge, suggesting enhanced repulsive electrostatic interactions and, consequently, a stabilized colloidal system. These results show that electrophoretic mobility is a useful methodology that can be applied to screen the stabilization factors for virus-like particles during vaccine development.

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<![CDATA[Motion of Molecular Probes and Viscosity Scaling in Polyelectrolyte Solutions at Physiological Ionic Strength]]> https://www.researchpad.co/article/5989d9ebab0ee8fa60b6ca29

We investigate transport properties of model polyelectrolyte systems at physiological ionic strength (0.154 M). Covering a broad range of flow length scales—from diffusion of molecular probes to macroscopic viscous flow—we establish a single, continuous function describing the scale dependent viscosity of high-salt polyelectrolyte solutions. The data are consistent with the model developed previously for electrically neutral polymers in a good solvent. The presented approach merges the power-law scaling concepts of de Gennes with the idea of exponential length scale dependence of effective viscosity in complex liquids. The result is a simple and applicable description of transport properties of high-salt polyelectrolyte solutions at all length scales, valid for motion of single molecules as well as macroscopic flow of the complex liquid.

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<![CDATA[Role of non-native electrostatic interactions in the coupled folding and binding of PUMA with Mcl-1]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf411

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit DebyeHückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process. This model was carefully calibrated with the experimental data on helical content and affinity, and shown to be consistent with previously published experimental data on binding rate changes with respect to ionic strength. We find that intramolecular electrostatic interactions influence the unbound states of PUMA only marginally. Our results further suggest that intermolecular electrostatic interactions, and in particular non-native electrostatic interactions, are involved in formation of the initial encounter complex. We are able to reveal the binding mechanism in more detail than is possible using experimental data alone however, and in particular we uncover the role of non-native electrostatic interactions. We highlight the potential importance of such electrostatic interactions for describing the binding reactions of IDPs. Such approaches could be used to provide predictions for the results of mutational studies.

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<![CDATA[Towards a critical evaluation of an empirical and volume-based solvation function for ligand docking]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc0e8

Molecular docking is an important tool for the discovery of new biologically active molecules given that the receptor structure is known. An excellent environment for the development of new methods and improvement of the current methods is being provided by the rapid growth in the number of proteins with known structure. The evaluation of the solvation energies outstands among the challenges for the modeling of the receptor-ligand interactions, especially in the context of molecular docking where a fast, though accurate, evaluation is ought to be achieved. Here we evaluated a variation of the desolvation energy model proposed by Stouten (Stouten P.F.W. et al, Molecular Simulation, 1993, 10: 97–120), or SV model. The SV model showed a linear correlation with experimentally determined solvation energies, as available in the database FreeSolv. However, when used in retrospective docking simulations using the benchmarks DUD, charged-matched DUD and DUD-Enhanced, the SV model resulted in poorer enrichments when compared to a pure force field model with no correction for solvation effects. The data provided here is consistent with other empirical solvation models employed in the context of molecular docking and indicates that a good model to account for solvent effects is still a goal to achieve. On the other hand, despite the inability to improve the enrichment of retrospective simulations, the SV solvation model showed an interesting ability to reduce the number of molecules with net charge -2 and -3 e among the top-scored molecules in a prospective test.

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<![CDATA[The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor]]> https://www.researchpad.co/article/5989d9feab0ee8fa60b73113

Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

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<![CDATA[Sampling and detection of airborne influenza virus towards point-of-care applications]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc1d7

Airborne transmission of the influenza virus contributes significantly to the spread of this infectious pathogen, particularly over large distances when carried by aerosol droplets with long survival times. Efficient sampling of virus-loaded aerosol in combination with a low limit of detection of the collected virus could enable rapid and early detection of airborne influenza virus at the point-of-care setting. Here, we demonstrate a successful sampling and detection of airborne influenza virus using a system specifically developed for such applications. Our system consists of a custom-made electrostatic precipitation (ESP)-based bioaerosol sampler that is coupled with downstream quantitative polymerase chain reaction (qPCR) analysis. Aerosolized viruses are sampled directly into a miniaturized collector with liquid volume of 150 μL, which constitutes a simple and direct interface with subsequent biological assays. This approach reduces sample dilution by at least one order of magnitude when compared to other liquid-based aerosol bio-samplers. Performance of our ESP-based sampler was evaluated using influenza virus-loaded sub-micron aerosols generated from both cultured and clinical samples. Despite the miniaturized collection volume, we demonstrate a collection efficiency of at least 10% and sensitive detection of a minimum of 3721 RNA copies. Furthermore, we show that an improved extraction protocol can allow viral recovery of down to 303 RNA copies and a maximum sampler collection efficiency of 47%. A device with such a performance would reduce sampling times dramatically, from a few hours with current sampling methods down to a couple of minutes with our ESP-based bioaerosol sampler.

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<![CDATA[Synthesis, characterization, and debromination reactivity of cellulose-stabilized Pd/Fe nanoparticles for 2,2',4,4'-tretrabromodiphenyl ether]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc875

In this study, two kinds of cellulose derivatives (polyanionic cellulose (PAC) and hydroxypropylmethyl cellulose (HPMC)) were selected as stabilizers of Pd/Fe nanoparticles (NPs) to investigate their influences on the debromination performances of 2,2',4,4'-tretrabromodiphenyl ether (BDE47). Field emission scanning electron microscope (FE-SEM) images revealed that the cellulose-stabilized Pd/Fe NPs were smaller and more uniform than the bare-Pd/Fe NPs. X-ray diffractometer (XRD) and X-ray photoelectron spectroscopy (XPS) results suggested that cellulose coatings found on Pd/Fe NPs surfaces featured some antioxidation abilities, which followed the order of HPMC < PAC. Sedimentation tests demonstrated that the stabilizing power of PAC for Pd/Fe NPs was higher than that of HPMC. Fourier transfer infrared spectrometer (FTIR) results indicated that PAC molecules were bound to the Pd/Fe NPs surfaces by polar covalent bonds and hydrogen bonds, while HPMC molecules interacted with the nanoparticles by hydrogen bonds. Batch debromination test for BDE47 demonstrated that the catalytic debromination rate with cellulose-stabilized Pd/Fe NPs was higher than that with bare-Pd/Fe NPs during reaction period of 15 min. Overall, this study indicated that both celluloses are beneficial to forming smaller, more regular, stable and antioxidative Pd/Fe NPs, leading to higher debromination reactivity for BDE47 compared with the bare-Pd/Fe NPs. Therefore Pd/Fe NPs can be utilized as a promising remediation technology for the contaminated groundwater and soils.

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<![CDATA[Variations in the interaction of human defensins with Escherichia coli: Possible implications in bacterial killing]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc8d5

Human α and β-defensins are cationic antimicrobial peptides characterized by three disulfide bonds with a triple stranded β-sheet motif. It is presumed that interaction with the bacterial cell surface and membrane permeabilization by defensins is an important step in the killing process. In this study, we have compared interactions of three human α-defensins HNP3, HNP4, HD5 and human β-defensins HBD1-4 that are active against Escherichia coli, with its cell surface and inner membrane as well as negatively charged model membranes. We have also included the inactive α-defensin HD6 in the study. Among the α-defensins, HNP4, HD5 and HD6 were more effective in increasing the zeta potential as compared to HNP3. Among the β-defensins, HBD1 was the least effective in increasing the zeta potential. The zeta potential modulation data indicate variations in the surface charge neutralizing ability of α- and β-defensins. Comparison of E. coli inner membrane and model membrane permeabilizing abilities indicated that HD5, HD6 and HBD1 do not permeabilize membranes. Although HBD4 does not permeabilize model membranes, considerable damage to the inner membrane of E. coli is observed. Our data indicate that mammalian defensins do not kill E. coli by a simple mechanism involving membrane permeabilization though their antibacterial potencies are very similar.

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<![CDATA[Induced Fit in Protein Multimerization: The HFBI Case]]> https://www.researchpad.co/article/5989d9e4ab0ee8fa60b6a967

Hydrophobins, produced by filamentous fungi, are small amphipathic proteins whose biological functions rely on their unique surface-activity properties. Understanding the mechanistic details of the multimerization process is of primary importance to clarify the interfacial activity of hydrophobins. We used free energy calculations to study the role of a flexible β-hairpin in the multimerization process in hydrophobin II from Trichoderma reesei (HFBI). We characterized how the displacement of this β-hairpin controls the stability of the monomers/dimers/tetramers in solution. The regulation of the oligomerization equilibrium of HFBI will necessarily affect its interfacial properties, fundamental for its biological function and for technological applications. Moreover, we propose possible routes for the multimerization process of HFBI in solution. This is the first case where a mechanism by which a flexible loop flanking a rigid patch controls the protein-protein binding equilibrium, already known for proteins with charged binding hot-spots, is described within a hydrophobic patch.

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<![CDATA[D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function]]> https://www.researchpad.co/article/5989daaeab0ee8fa60baa8b6

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.

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<![CDATA[Unusual Salt and pH Induced Changes in Polyethylenimine Solutions]]> https://www.researchpad.co/article/5989da8bab0ee8fa60b9e1b1

Linear PEI is a cationic polymer commonly used for complexing DNA into nanoparticles for cell-transfection and gene-therapy applications. The polymer has closely-spaced amines with weak-base protonation capacity, and a hydrophobic backbone that is kept unaggregated by intra-chain repulsion. As a result, in solution PEI exhibits multiple buffering mechanisms, and polyelectrolyte states that shift between aggregated and free forms. We studied the interplay between the aggregation and protonation behavior of 2.5 kDa linear PEI by pH probing, vapor pressure osmometry, dynamic light scattering, and ninhydrin assay. Our results indicate that:

  1. At neutral pH, the PEI chains are associated and the addition of NaCl initially reduces and then increases the extent of association.

  2. The aggregate form is uncollapsed and co-exists with the free chains.

  3. PEI buffering occurs due to continuous or discontinuous charging between stalled states.

  4. Ninhydrin assay tracks the number of unprotonated amines in PEI.

  5. The size of PEI-DNA complexes is not significantly affected by the free vs. aggregated state of the PEI polymer.

Despite its simple chemical structure, linear PEI displays intricate solution dynamics, which can be harnessed for environment-sensitive biomaterials and for overcoming current challenges with DNA delivery.

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<![CDATA[Structural analysis of P. falciparum KAHRP and PfEMP1 complexes with host erythrocyte spectrin suggests a model for cytoadherent knob protrusions]]> https://www.researchpad.co/article/5aafc0ef463d7e7cbd9135c1

Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) and Knob-associated Histidine-rich Protein (KAHRP) are directly linked to malaria pathology. PfEMP1 and KAHRP cluster on protrusions (knobs) on the P. falciparum-infected erythrocyte surface and enable pathogenic cytoadherence of infected erythrocytes to the host microvasculature, leading to restricted blood flow, oxygen deprivation and damage of tissues. Here we characterize the interactions of PfEMP1 and KAHRP with host erythrocyte spectrin using biophysical, structural and computational approaches. These interactions assist knob formation and, thus, promote cytoadherence. We show that the folded core of the PfEMP1 cytosolic domain interacts broadly with erythrocyte spectrin but shows weak, residue-specific preference for domain 17 of α spectrin, which is proximal to the erythrocyte cytoskeletal junction. In contrast, a protein sequence repeat region in KAHRP preferentially associates with domains 10–14 of β spectrin, proximal to the spectrin–ankyrin complex. Structural models of PfEMP1 and KAHRP with spectrin combined with previous microscopy and protein interaction data suggest a model for knob architecture.

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<![CDATA[Theory on the Mechanism of DNA Renaturation: Stochastic Nucleation and Zipping]]> https://www.researchpad.co/article/5989daefab0ee8fa60bc0acb

Renaturation of the complementary single strands of DNA is one of the important processes that requires better understanding in the view of molecular biology and biological physics. Here we develop a stochastic dynamical model on the DNA renaturation. According to our model there are at least three steps in the renaturation process viz. nonspecific-contact formation, correct-contact formation and nucleation, and zipping. Most of the earlier two-state models combined nucleation with nonspecific-contact formation step. In our model we suggest that it is considerably meaningful when we combine the nucleation with the zipping since nucleation is the initial step of zipping and nucleated and zipping molecules are indistinguishable. Nonspecific contact formation step is a pure three-dimensional diffusion controlled collision process. Whereas nucleation involves several rounds of one-dimensional slithering and internal displacement dynamics of one single strand of DNA on the other complementary strand in the process of searching for the correct-contact and then initiate nucleation. Upon nucleation, the stochastic zipping follows to generate a fully renatured double stranded DNA. It seems that the square-root dependency of the overall renaturation rate constant on the length of reacting single strands originates mainly from the geometric constraints in the diffusion controlled nonspecific-contact formation step. Further the inverse scaling of the renaturation rate on the viscosity of reaction medium also originates from nonspecific contact formation step. On the other hand the inverse scaling of the renaturation rate with the sequence complexity originates from the stochastic zipping which involves several rounds of crossing over the free-energy barrier at microscopic levels. When the sequence of renaturing single strands of DNA is repetitive with less complexity then the cooperative effects will not be noticeable since the parallel zipping will be a dominant enhancing factor. However for DNA strands with high sequence complexity and length one needs to consider the underlying cooperative effects both at microscopic and macroscopic levels to explain various scaling behaviours of the overall renaturation rate.

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<![CDATA[The Effects of Ca2+ Concentration and E200K Mutation on the Aggregation Propensity of PrPC: A Computational Study]]> https://www.researchpad.co/article/5989daa5ab0ee8fa60ba7299

The propensity of cellular prion protein to aggregation is reputed essential for the initiation of the amyloid cascade that ultimately lead to the accumulation of neurotoxic aggregates. In this paper, we extended and applied an already reported computational workflow [Proteins 2015; 83: 1751–1765] to elucidate in details the aggregation propensity of PrP protein systems including wild type, wild type treated at different [Ca2+] and E200K mutant. The application of the computational procedure to two segments of PrPC, i.e. 125–228 and 120–231, allowed to emphasize how the inclusion of complete C-terminus and last portion (120–126) of the neurotoxic segment 106–126 may be crucial to unveil significant and unexpected interaction properties. Indeed, the anchoring of N-terminus on H2 domain detected in the wild type resulted to be disrupted upon either E200K mutation or Ca2+ binding, and to unbury hydrophobic spots on the PrPC surface. A peculiar dinuclear Ca2+ binding motif formed by the C-terminus and the S2-H2 loop was detected for [Ca2+] > 5 mM and showed similarities with binding motifs retraced in other protein systems, thus suggesting a possible functional meaning for its formation. Therefore, we potentiated the computational procedure by including a tool that clusterize the minima of molecular interaction fields of a proteinand delimit the regions of space with higher hydrophobic or higher hydrophilic character, hence, more likely involved in the self-assembly process. Plausible models for the self-assembly of either the E200K mutated or Ca2+-bound PrPC were sketched and discussed. The present investigation provides for structure-based information and new prompts that may represent a starting point for future experimental or computational works on the PrPC aggregation.

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