Scalable Nanohelices for Predictive Studies and Enhanced 3D Visualization

Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing applications.  For predictive simulations, it has become increasingly important to be able to model the structure of nanohelices accurately.  To study the effect of local structure on the properties of these complex geometries one must develop realistic models.  To date, software packages are rather limited in creating atomistic helical models.  This work focuses on producing atomistic models of silica glass (SiO₂) nanoribbons and nanosprings for molecular dynamics (MD) simulations. Using an MD model of “bulk” silica glass, two computational procedures to precisely create the shape of nanoribbons and nanosprings are presented.  The first method employs the AWK programming language and open-source software to effectively carve various shapes of silica nanoribbons from the initial bulk model, using desired dimensions and parametric equations to define a helix.  With this method, accurate atomistic silica nanoribbons can be generated for a range of pitch values and dimensions.  The second method involves a more robust code which allows flexibility in modeling nanohelical structures.  This approach utilizes a C++ code particularly written to implement pre-screening methods as well as the mathematical equations for a helix, resulting in greater precision and efficiency when creating nanospring models.  Using these codes, well-defined and scalable nanoribbons and nanosprings suited for atomistic simulations can be effectively created.  An added value in both open-source codes is that they can be adapted to reproduce different helical structures, independent of material.  In addition, a MATLAB graphical user interface (GUI) is used to enhance learning through visualization and interaction for a general user with the atomistic helical structures.  One application of these methods is the recent study of nanohelices via MD simulations for mechanical energy harvesting purposes.     

Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel

Large-conductance Ca²⁺- and voltage-activated K⁺ channel (BK) open probability is enhanced by depolarization, increasing Ca²⁺ concentration, or both. These stimuli activate modular voltage and Ca²⁺ sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca²⁺, profoundly hinders channel opening while showing only minor effects on the voltage sensor active–resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca²⁺ binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open–closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open–closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Exploring QSAR for CYP11B2 binding affinity and CYP11B2/CYP11B1 selectivity of diverse functional compounds using GFA and G/PLS techniques

A data set of a series of 132 structurally diverse compounds with cytochrome 11B2 and 11B1 (CYP11B2 and CYP11B1) enzyme inhibitory activities was subjected to molecular shape analysis to explore contributions of shape features as well as electronic, structural, and physicochemical parameters toward enzyme inhibitory activities, in search of appropriate molecular scaffolds with optimum substitutions for highly potent CYP11B2 inhibitors. Genetic function approximation (GFA) and genetic partial least squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical quality considering both internal and external validation parameters (Q²: 0.514–0.659, R²_pred: 0.510–0.734). The G/PLS models with spline option for CYP11B2 and CYP11B1 inhibition and selectivity modeling appeared to be the best models based on r_m²_(overall) criterion. The study indicates the importance of the pyridinylnaphthalene and pyridylmethylene-indane scaffolds with less polar and electrophilic substituents for optimum CYP11B2 inhibitory activity and CYP11B2/CYP11B1 selectivity.     

Structural bioinformatics insights into the CARD-CARD interaction mediated by the mitochondrial antiviral-signaling protein of black carp

The innate immune system offers the first line of defense against invading microbial pathogens through the recognition of conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). The host innate immune system through PRRs, the sensors for PAMPs, induces the production of cytokines. Among different families of PRRs, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and its mitochondrial adaptor ie, the mitochondrial antiviral-signaling (MAVS) protein, are crucial for RLR-triggered interferon (IFN) antiviral immunity. Recent studies have shown that the N-terminal caspase recruitment domain (CARD) and transmembrane domain play a pivotal role in oligomerization of black carp MAVS (BcMAVS), crucial for the host innate immune response against viral invasion. In this study, we have used molecular modeling, docking, and molecular dynamics (MD) simulation approaches to shed molecular insights into the oligomerization mechanism of BcMAVS^CARD. MD simulation and interaction analysis portrayed that the type-I surface patches of BcMAVS ^CARD make the major contribution to the interaction. Moreover, the evidence from surface patches and critical residues involved in the said interaction is found to be similar to that of the human counterpart and requires further investigation for legitimacy. Altogether, our study provided crucial information on oligomerization of BcMAVS CARDs and might be helpful for clarifying the innate immune response against pathogens and downstream signaling in fishes.     

Design of novel quinoline-aminopiperidine derivatives as Mycobacterium tuberculosis (MTB) GyrB inhibitors: an in silico study

Tuberculosis (TB) is an infectious disease that causes a number of deaths, and the development of new, safer and more adequate TB inhibitors/drugs has become a necessity as well as a great challenge. Mycobacterial DNA gyrase B subunit has been identified to be one of the potentially underexploited drug targets in the field of anti-tubercular drug discovery. To design the novel and potent Mycobacterium tuberculosis (MTB) inhibitors, we performed molecular modeling studies that combined the 3D-QSAR, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. Forty eight quinoline-aminopiperidine inhibitors which act on DNA gyrase B subunit were used for constructing 3D-QSAR models. The results showed that the best CoMFA model had the high performance with q²?=?0.643, r²?=?0.947, while the best CoMSIA model yielded q²?=?0.536, r²?=?0.948. The contour map was in good agreement with the docking and MD simulations which strongly demonstrated that the molecular modeling was reliable. Based on this information, several potential compounds were designed and their inhibitory activities were also verified by the accomplished models and ADME/T predictions. We hope that our research could bring new ideas to facilitate the development of novel inhibitors with higher inhibitory activity for TB.

Communicated by Ramaswamy H. Sarma     

Designing of MIP based QCM sensor having thymine recognition sites based on biomimicking DNA approach

Quartz crystal microbalance (QCM) sensors coated with molecular imprinted polymers (MIP) have been developed for the determination of thymine. In this method, methacryloylamidoadenine (MA-Ade) have used as a new monomer and thymine template for inspiration of DNA nucleobases interaction. The thymine can be simultaneously hydrogen binding to MA-Ade and fit into the shape-selective cavities. Thus, the interaction between nucleobases has an effect on the binding ability of the QCM sensors. The binding affinity of the thymine imprinted sensors has investigated by using the Langmuir isotherm. The thymine imprinted QCM electrodes have shown homogeneous binding sites for thymine (K_a: 1.0 × 10⁵ M⁻¹) while heterogeneous binding sites for uracil. On the other hand, recognition selectivity of the QCM sensor based on thymine imprinted polymer toward to uracil, ssDNA and ssRNA has been reported in this work.     

A sensitive SERS quantitative analysis method for Ni2+ by the dimethylglyoxime reaction regulating a graphene oxide nanoribbon catalytic gold nanoreaction

The nanogold reaction between HAuCl₄ and trisodium citrate (TCA) proceeded very slowly at 60°C in a water bath. The as‐prepared graphene oxide nanoribbons (GONRs) exhibited strong catalysis during the reaction to form gold nanoparticles (Au NPs) and appeared as a strong surface‐enhanced Raman scattering (SERS) peak at 1616 cm^?1 in the presence of the molecular probe Victoria blue 4R (VB4r). With increase in GONR concentration, the SERS peak increased due to increased formation of Au NPs. Upon addition of dimethylglyoxime (DMG) ligand, which was adsorbed onto the GONR surface to inhibit GONR catalysis, the SERS peak decreased. When Ni²⁺ was added, a coordination reaction between DMG and Ni²⁺ took place to form stable complexes of [Ni (DMG)₂]²⁺ and the release of free GONR catalyst that resulted in the SERS peak increasing linearly. A SERS quantitative analysis method for Ni²⁺ was therefore established, with a linear range of 0.07–2.8 μM, and a detection limit of 0.036 μM Ni²⁺.     

Two Structural Motifs within Canonical EF-Hand Calcium-Binding Domains Identify Five Different Classes of Calcium Buffers and Sensors

Proteins with EF-hand calcium-binding motifs are essential for many cellular processes, but are also associated with cancer, autism, cardiac arrhythmias, and Alzheimer''s, skeletal muscle and neuronal diseases. Functionally, all EF-hand proteins are divided into two groups: (1) calcium sensors, which function to translate the signal to various responses; and (2) calcium buffers, which control the level of free Ca²⁺ ions in the cytoplasm. The borderline between the two groups is not clear, and many proteins cannot be described as definitive buffers or sensors. Here, we describe two highly-conserved structural motifs found in all known different families of the EF-hand proteins. The two motifs provide a supporting scaffold for the DxDxDG calcium binding loop and contribute to the hydrophobic core of the EF hand domain. The motifs allow more precise identification of calcium buffers and calcium sensors. Based on the characteristics of the two motifs, we could classify individual EF-hand domains into five groups: (1) Open static; (2) Closed static; (3) Local dynamic; (4) Dynamic; and (5) Local static EF-hand domains.     

Using multiple satellite observations to quantitatively assess and model oil pollution and predict risks and consequences to shoreline from oil platforms in the Caspian Sea

This research focused on the following objectives: (1) using satellite data to characterize the spatiotemporal distribution of anthropogenic oil spills from Oil Rocks Settlement, Chilov and Pirallahi Islands (2) stochastic modeling of the oil spill risk pose to water quality and shoreline ecosystems, and (3) validating model predictions using satellite images. 165 satellite images acquired by SENTINEL-1A, LANDSAT-8, RADARSAT, ENVISAT and ERS sensors between 1996 and 2015 were used for the detection of oil spills using object-based classification and visual interpretation. Anthropogenic hotspots were observed at three oldest oil production sites with estimated oil spilling up to 1264 m³ per day and different degrees of temporal repetition of oil spills. The largest area (5639 km²) experienced 1–10 detected oil spills, while 993 km² experienced 11–20 oil spills, 775 km² experienced 21–50 oil spills, 208 km² experienced 51–100 oil spills, and 36 km² experienced 101–150 oil spills. The majority (83% or 6157 km²) of sea surface area within the combined boundary of detected oil spills (7422 km²) had a 50% or greater chance of oil spill contamination. Approximately, 6% (44 km of 751 km) of shoreline had a 50% or greater probability of contamination with land use classes sensitive to pollution.     

The <Emphasis Type="Italic">Drosophila</Emphasis>STIM1 orthologue,dSTIM, has roles in cell fate specification and tissue patterning

Background  

Mammalian STIM1 and STIM2 and the single Drosophila homologue dSTIM have been identified as key regulators of store-operated Ca²⁺ entry in cells. STIM proteins function both as molecular sensors of Ca²⁺concentration in the endoplasmic reticulum (ER) and the molecular triggers that activate SOC channels in the plasma membrane. Ca²⁺ is a crucial intracellular messenger utilised in many cellular processes, and regulators of Ca²⁺ homeostasis in the ER and cytosol are likely to play important roles in developmental processes. STIM protein expression is altered in several tumour types but the role of these proteins in developmental signalling pathways has not been thoroughly examined.     

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

Plant responses to developmental and environmental cues are often mediated by calcium (Ca²⁺) signals that are transmitted by diverse calcium sensors. The calcineurin B-like (CBL) protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases. We report functional analysis of Arabidopsis CBL2 and CBL3, two closely related CBL members that are localized to the vacuolar membrane through the N-terminal tonoplast-targeting sequence. While cbl2 or cbl3 single mutant did not show any phenotypic difference from the wild type, the cbl2 cbl3 double mutant was stunted with leaf tip necrosis, underdeveloped roots, shorter siliques and fewer seeds. These defects were reminiscent of those in the vha-a2 vha-a3 double mutant deficient in vacuolar H⁺-ATPase (V-ATPase). Indeed, the V-ATPase activity was reduced in the cbl2 cbl3 double mutant, connecting tonoplast CBL-type calcium sensors to the regulation of V-ATPase. Furthermore, cbl2 cbl3 double mutant was compromised in ionic tolerance and micronutrient accumulation, consistent with the defect in V-ATPase activity that has been shown to function in ion compartmentalization. Our results suggest that calcium sensors CBL2 and CBL3 serve as molecular links between calcium signaling and V-ATPase, a central regulator of intracellular ion homeostasis.     

Calcium decoding mechanisms in plants

Ca²⁺ is a crucial second messenger that is involved in mediating responses to various biotic and abiotic environmental cues and in the regulation of many developmental processes in plants. Intracellular Ca²⁺ signals are realized by spatially and temporally defined changes in Ca²⁺ concentration that represent stimulus-specific Ca²⁺ signatures. These Ca²⁺ signatures are sensed, decoded and transmitted to downstream responses by a complex tool kit of Ca²⁺ binding proteins that function as Ca²⁺ sensors. Plants possess an extensive and complex array of such Ca²⁺ sensors that convey the information presented in the Ca²⁺ signatures into phosphorylation events, changes in protein-protein interactions or regulation of gene expression. Prominent Ca²⁺ sensors like, Calmodulins (CaM), Calmodulin-like proteins (CMLs), calcium dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs) exist in complex gene families and form intricate signaling networks in plants that are capable of robust and flexible information processing. In this review we reflect on the recently gained knowledge about the mechanistic principles of these Ca²⁺ sensors, their biochemical properties, physiological functions and newly identified targets proteins. These aspects will be discussed in the context of emerging functional principles that govern the information processing via these signaling modules.     

A novel rice calmodulin-like gene, <Emphasis Type="Italic">OsMSR2,</Emphasis> enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis

Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca²⁺ signals are perceived by different Ca²⁺ sensors, and calmodulin (CaM) is one of the best-characterized Ca²⁺ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei’ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca²⁺ in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.     

Activation and regulation of store‐operated calcium entry

The process of store-operated Ca²⁺ entry (SOCE), whereby Ca²⁺ influx across the plasma membrane is activated in response to depletion of intracellular Ca²⁺ stores in the endoplasmic reticulum (ER), has been under investigation for greater than 25 years; however, only in the past 5 years have we come to understand this mechanism at the molecular level. A surge of recent experimentation indicates that STIM molecules function as Ca²⁺ sensors within the ER that, upon Ca²⁺ store depletion, rearrange to sites very near to the plasma membrane. At these plasma membrane-ER junctions, STIM interacts with and activates SOCE channels of the Orai family. The molecular and biophysical data that have led to these findings are discussed in this review, as are several controversies within this rapidly expanding field.     

Conformational analysis of CCK-B agonists using 1H-nmr and restrained molecular dynamics: Comparison of biologically active Boc-Trp-(N-Me)Nle-Asp-Phe-NH2 and inactive Boc-Trp-(N-Me)Phe-Asp-Phe-NH2

The tetrapeptide Boc-Trp-(N-Me)Nle-Asp-Phe-NH₂ is a potent CCK-B agonist. Replacement in this analogue of the norleucine residue by a phenylalanine, to yield Boc-Trp-(N-Me)Phe-Asp-Phe-NH₂, led to a 740-fold decrease in affinity whereas the same decrease in affinity was not observed in their nonmethylated counterparts. In order to ascertain the conformational preferences of these two N-methylated tetrapeptides, a study by two-dimensional (2D) nmr spectroscopy and molecular modeling was undertaken. The solution conformation of the two peptides was examined by ¹H-nmr in a d₆-DMSO/H₂O (80 : 20) mixture. A cis-trans equilibrium, induced by N-methylation, was observed for both analogues, and the proton spectra of the two retamers were fully characterized in each case. ¹H-¹H distance constraints, derived from 2D nuclear Overhauser effect spectroscopy and rotating frame nuclear Overhauser effect spectroscopy experiments, were used as inputs for subsequent restrained molecular dynamics simulations. Comparisons of the nmr and molecular modeling data point toward distinct conformational preferences for these two peptides with an opposite spatial orientation of the Trp residue, and could explain the large difference in their biological activities. Furthermore, the tridimensional structure of Boc-Trp-(N-Me)Nle-Asp-Phe-NH₂ could serve as a model for the design of nonpeptide CCK-B agonists. © 1994 John Wiley & Sons, Inc.     

Functional and Modeling Studies of the Transmembrane Region of the TRPM8 Channel

Members of the transient receptor potential (TRP) ion channel family act as polymodal cellular sensors, which aid in regulating Ca²⁺ homeostasis. Within the TRP family, TRPM8 is the cold receptor that forms a nonselective homotetrameric cation channel. In the absence of TRPM8 crystal structure, little is known about the relationship between structure and function. Inferences of TRPM8 structure have come from mutagenesis experiments coupled to electrophysiology, mainly regarding the fourth transmembrane helix (S4), which constitutes a moderate voltage-sensing domain, and about cold sensor and phosphatidylinositol 4,5-bisphosphate binding sites, which are both located in the C-terminus of TRPM8. In this study, we use a combination of molecular modeling and experimental techniques to examine the structure of the TRPM8 transmembrane and pore helix region including the conducting conformation of the selectivity filter. The model is consistent with a large amount of functional data and was further tested by mutagenesis. We present structural insight into the role of residues involved in intra- and intersubunit interactions and their link with the channel activity, sensitivity to icilin, menthol and cold, and impact on channel oligomerization.     

Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature

Background

Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).

Results

Here we report significant progress on the development of the latter type of Ca²⁺ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺ response to a prolonged glutamate treatment in cortical neurons.

Conclusion

We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.     

Discovery of FIXa inhibitors by combination of pharmacophore modeling,molecular docking,and 3D-QSAR modeling

Human Coagulation Factor IXa (FIXa), specifically inhibited at the initiation stage of the blood coagulation cascade, is an excellent target for developing selective and safe anticoagulants. To explore this inhibitory mechanism, 86 FIXa inhibitors were selected to generate pharmacophore models and subsequently SAR models. Both best pharmacophore model and ROC curve were built through the Receptor–Ligand Pharmacophore Generation module. CoMFA model based on molecular docking and PLS factor analysis methods were developed. Model propagations values are q²?=?0.709, r²?=?0.949, and r²_pred?=?0.905. The satisfactory q² value of 0.609, r² value of 0.962, and r²_pred value of 0.819 for CoMSIA indicated that the CoMFA and CoMSIA models are both available to predict the inhibitory activity on FIXa. On the basis of pharmacophore modeling, molecular docking, and 3D-QSAR modeling screening, six molecules are screened as potential FIXa inhibitors.