Buried Sites Research Articles

Interfacial Water Many-Body Effects Drive Structural Dynamics and Allosteric Interactions in SARS-CoV-2 Main Protease Dimerization Interface.

Following our previous work (Chem. Sci.2021, 12, 4889−490734168762), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 μs) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is stable only at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy because they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physicochemical microenvironments. The water–interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.

CVD Polycrystalline Graphene as Sensing Film of Extended-Gate ISFET for Low-Drift pH Sensor

Drift phenomenon is a critical issue which causes reading errors in an ion sensitive field effect transistor (ISFET). The gate insulator surface modifications, such as hydration and buried site formation, which result from the diffusion of water species from the electrolyte, play the key roles in drift phenomenon. Here, the use of two-dimensional (2D) chemical vapor deposited (CVD) graphene as the sensing film of ISFETs to reduce drift has been proposed and investigated. Density functional theory calculations indicate good impermeability of the 2D graphene layer against water molecules, which may resist water species diffusion and contribute to low drift performance. The experimental measurements have shown that graphene acting as the extended-gate sensing film of ISFET exhibits a small drift rate of about 0.4 mV h−1 with a pH sensitivity of 33 mV pH−1. The almost drift-free performance was measured immediately after device fabrication without any pre-immersion processes, indicating a superior stability of 2D CVD graphene.

THE SEISMIC BEHAVIOR OF BURIED SEABED WALLS IN LIQUEFACTION SOIL


 
 
 The present study aimed to investigate the seismic behavior of enclosed seawater walls, the buried site of which lies in liquefaction soil. An experimental specimen was manufactured and tested on the seismic table, and a numerical study was also modeled in the ABAQUS software based on the experimental outcomes. In both the experimental and numerical studies, a susceptible liquefaction layer around the root of the wall was considered due to the root lean soil leakage and large lateral pressure, and the soil behind the root caused the failure of the buried section. According to the results, the lateral movement significantly decreased due to the backing effect of this layer on the buried section of the wall. Furthermore, an active wedge was formed from the buried side to the back of the containment, and the braces were overwhelming due to the presence of the locks in the wedge and their movement along with the wedge. The displacement of the crown and foot of the wall decreased with the increased base acceleration and higher frequency of the entrance movement.
 
 

The Roles of Protein Structure, Taxon Sampling, and Model Complexity in Phylogenomics: A Case Study Focused on Early Animal Divergences

Despite the long history of using protein sequences to infer the tree of life, the potential for different parts of protein structures to retain historical signal remains unclear. We propose that it might be possible to improve analyses of phylogenomic datasets by incorporating information about protein structure. We test this idea using the position of the root of Metazoa (animals) as a model system. We examined the distribution of “strongly decisive” sites (alignment positions that support a specific tree topology) in a dataset comprising >1500 proteins and almost 100 taxa. The proportion of each class of strongly decisive sites in different structural environments was very sensitive to the model used to analyze the data when a limited number of taxa were used but they were stable when taxa were added. As long as enough taxa were analyzed, sites in all structural environments supported the same topology regardless of whether standard tree searches or decisive sites were used to select the optimal tree. However, the use of decisive sites revealed a difference between the support for minority topologies for sites in different structural environments: buried sites and sites in sheet and coil environments exhibited equal support for the minority topologies, whereas solvent-exposed and helix sites had unequal numbers of sites, supporting the minority topologies. This suggests that the relatively slowly evolving buried, sheet, and coil sites are giving an accurate picture of the true species tree and the amount of conflict among gene trees. Taken as a whole, this study indicates that phylogenetic analyses using sites in different structural environments can yield different topologies for the deepest branches in the animal tree of life and that analyzing larger numbers of taxa eliminates this conflict. More broadly, our results highlight the desirability of incorporating information about protein structure into phylogenomic analyses.

Unveiling the “invisible” druggable conformations of GDP-bound inactive Ras

The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale. The experimentally informed ensemble unequivocally demonstrates the preformation of both surface-exposed and buried cryptic sites in Ras•GDP, advocating design of inhibition by targeting the transient druggable conformers that are invisible to conventional experimental methods. The viability of the ensemble-based rational design has been established by retrospective testing of the ability of the Ras•GDP ensemble to identify known ligands from decoys in virtual screening.

Resurrected Ancestral TIM-Barrel Glycosidase Displays Heme Binding and Allosteric Modulation

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. In this work, we report an ancestral sequence reconstruction exercise targeting ancient nodes during the evolution of family-1 glycosidases and present the exceptional properties of a putative resurrected ancestor of bacterial and eukaryotic enzymes. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures for ∼1400 modern glycosidases show a porphyrin ring in the enzyme. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein resurrection to reveal valuable but unusual protein features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

A Buried Middle Archaic Site in the Tucson Basin

Despite great gains in knowledge about the Late Archaic/Early Agricultural period in the Tucson Basin, understanding of the Middle Archaic period remains slim. This paper reports on volunteer investigations at three buried Archaic sites within the Santa Cruz River floodplain: the Joe Ben Site (AZ BB:13:11), AZ BB:13:70, and an unnumbered locus north of the latter. The AZ BB:13:70 artifact assemblage and the Joe Ben site records suggest small-scale, short-term, repeated use of this portion of the Santa Cruz floodplain and the Santa Rita bajada by foragers between 4200 and 5100 BP, continuing into the Late Archaic/Early Agricultural period. Though assumed to be absent within the southern Southwest during the Middle and Late Holocene, the skull of a juvenile bison at AZ BB:13:70 and post-cranial bison bones at the unnumbered locus indicate their sporadic presence within the Tucson Basin during this period and their occasional exploitation by Middle Archaic hunters.

The molecular basis for the pH-dependent calcium affinity of the pattern recognition receptor langerin

The C-type lectin receptor langerin plays a vital role in the mammalian defense against invading pathogens. Langerin requires a Ca2+ cofactor, the binding affinity of which is regulated by pH. Thus, Ca2+ is bound when langerin is on the membrane but released when langerin and its pathogen substrate traffic to the acidic endosome, allowing the substrate to be degraded. The change in pH is sensed by protonation of the allosteric pH sensor histidine H294. However, the mechanism by which Ca2+ is released from the buried binding site is not clear. We studied the structural consequences of protonating H294 by molecular dynamics simulations (total simulation time: about 120 μs) and Markov models. We discovered a relay mechanism in which a proton is moved into the vicinity of the Ca2+-binding site without transferring the initial proton from H294. Protonation of H294 unlocks a conformation in which a protonated lysine side chain forms a hydrogen bond with a Ca2+-coordinating aspartic acid. This destabilizes Ca2+ in the binding pocket, which we probed by steered molecular dynamics. After Ca2+ release, the proton is likely transferred to the aspartic acid and stabilized by a dyad with a nearby glutamic acid, triggering a conformational transition and thus preventing Ca2+ rebinding. These results show how pH regulation of a buried orthosteric binding site from a solvent-exposed allosteric pH sensor can be realized by information transfer through a specific chain of conformational arrangements.

Polyaniline dispersed by Kevlar fiber for uptake of organic dye

PurposeThis paper aims to focus on the preparation of Kevlar fiber (KF) and alkaline hydrolyzed KF (KF-H) to improve the dispersed condition of polyaniline (PAn), as the aggregation of PAn would lead to some adsorption sites buried. And then the materials were used to enrich anionic dye Congo red (CR) from aqueous solution.Design/methodology/approachThe materials (KF@PAn and KF-H@PAn) were designed by means of “diffusion-interfacial-polymerization” under mild condition as high affinity due to the structural properties of PAn, KF and KF-H. The dispersed degree of PAn on the surface of KF and KF-H was validated according to adsorption efficiency for CR.FindingsThe content of PAn introduced was not beyond 20 wt.%, while adsorption capacity for CR was significantly enhanced by 4–8 times (on the basis of kinetic data) according to the calculation only by the content of PAn due to KF and alkaline hydrolyzed KF exhibited almost no adsorption for CR, indicating dispersed situation of PAn coating was greatly enhanced and more active sites exposed, which was favorable for the adsorption process. Presence of NaCl would exhibit a more or less positive effect on CR uptake, suggesting the materials could be used for high salt environment.Research limitations/implicationsThe investigated means of dispersed degree of PAn on the surface of KF and KF-H are the further and future investigation.Practical implicationsThis study will provide a method to improve the dispersed situation of PAn and a theoretical support to treat anionic dyes from aqueous solution especially for salt environment.Originality/valueThe results showed that the dispersed condition of PAn on the surface of KF and KF-H was greatly improved. According to the adsorption capacities for CR, it can be concluded that part of adsorption sites were buried due to the aggregation of PAn, and introduction of KF and KF-H, buried adsorption sites decreased greatly. This study will provide a method to decrease buried adsorption sites of PAn and a contribution for their convenient application in wastewater treatment especially for high salt environment.

Charge-Changing Perturbations and Path Sampling via Classical Molecular Dynamic Simulations of Simple Guest-Host Systems.

Currently, two different methods dominate the field of biomolecular free-energy calculations for the prediction of binding affinities. Pathway methods are frequently used for large ligands that bind on the surface of a host, such as protein–protein complexes. Alchemical methods, on the other hand, are preferably applied for small ligands that bind to deeply buried binding sites. The latter methods are also widely known to be heavily artifacted by the representation of electrostatic energies in periodic simulation boxes, in particular, when net-charge changes are involved. Different methods have been described to deal with these artifacts, including postsimulation correction schemes and instantaneous correction schemes (e.g., co-alchemical perturbation of ions). Here, we use very simple test systems to show that instantaneous correction schemes with no change in the system net charge lower the artifacts but do not eliminate them. Furthermore, we show that free energies from pathway methods suffer from the same artifacts.

Kinetics and Mechanism Comparison between Cr/Ti‐Based Bimetallic and Ti‐Based Monometallic Catalysts for Ethylene Polymerization

AbstractTwo (SiO2/MgR2/MgCl2)·TiClx model catalysts are made by refluxing TiCl4 with 0.35 wt% Cr modified silica gel/alkyl Mg adducts or silica gel/alkyl Mg adducts, which are named as Cr/Ti‐based bimetallic Cat‐1 and Ti‐based monometallic Cat‐2, respectively. The kinetics, active center counting, morphology, and polymer characterizations are studied to disclose the effect of low loading Cr active sites on the Cr/Ti‐based bimetallic Cat‐1 polymerization under mild conditions. The activity of Cat‐1 is 120.4% higher than that of Cat‐2, with a 114.1% higher [C*]/[M] value. Morphology results show the Cat‐1 fragmentation in the first 3 min is highly accelerated, which helps to release buried clustered Ti sites. Differential scanning calorimetry results show that low‐temperature heat absorbing shoulder of polyethylene (PE) from Cat‐2 demonstrates the signal of low crystallinity polymer made by Cat‐2 during the first 60 s, verifying the fluffy polymer in morphology results. GPC results show PE from Cat‐1 has a higher Mw in the first 3 min while a lower Mw in the end. The Cat‐1, which release active sites faster, has a high Mw in the early time. Lower Mw in the 900 s attributes to the effect of relative lower Mw polymer made by Cr sites, compared with Cat‐2.

Enhancing water sampling of buried binding sites using nonequilibrium candidate Monte Carlo.

Water molecules can be found interacting with the surface and within cavities in proteins. However, water exchange between bulk and buried hydration sites can be slow compared to simulation timescales, thus leading to the inefficient sampling of the locations of water. This can pose problems for free energy calculations for computer-aided drug design. Here, we apply a hybrid method that combines nonequilibrium candidate Monte Carlo (NCMC) simulations and molecular dynamics (MD) to enhance sampling of water in specific areas of a system, such as the binding site of a protein. Our approach uses NCMC to gradually remove interactions between a selected water molecule and its environment, then translates the water to a new region, before turning the interactions back on. This approach of gradual removal of interactions, followed by a move and then reintroduction of interactions, allows the environment to relax in response to the proposed water translation, improving acceptance of moves and thereby accelerating water exchange and sampling. We validate this approach on several test systems including the ligand-bound MUP-1 and HSP90 proteins with buried crystallographic waters removed. We show that our BLUES (NCMC/MD) method enhances water sampling relative to normal MD when applied to these systems. Thus, this approach provides a strategy to improve water sampling in molecular simulations which may be useful in practical applications in drug discovery and biomolecular design.

Diazonium Electrografting vs. Physical Adsorption of Azure A at Carbon Nanotubes for Mediated Glucose Oxidation with FAD‐GDH

AbstractThe electrochemical reduction of aryldiazonium salts is a versatile and direct route to obtain robust covalently modified electrodes. We report here a comparative study of Azure A modified carbon nanotube electrodes prepared by diazonium electrografting and by physical adsorption for bioelectrocatalytic glucose oxidation with fungal FAD‐glucose dehydrogenase from Aspergillus sp. The electrografted and adsorbed electrodes exhibited different reversible electroactivity consistent with polymer‐type and monomer‐type phenothiazine surface assemblies, respectively. The electrografted Azure A electrodes exhibited superior mediated bioelectrocatalysis compared to the adsorbed Azure A electrodes. A more than 10‐fold higher catalytic current up to 2 mA cm−2 at 0.2 V vs. Ag/AgCl together with a similarly low onset potential of −0.05 V vs. Ag/AgCl was observed at the electrografted electrodes. Faster estimated electron transfer kinetics and a +200 mV potential shift for the polymer‐type redox couple vs. the adsorbed monomer‐type couple underlines the favourable driving force for mediated electron transfer with the buried FAD active site for the diazonium‐derived bioelectrode.

Grand: A Python Module for Grand Canonical Water Sampling in OpenMM.

Networks of water molecules can play a critical role at the protein-ligand interface and can directly influence drug-target interactions. Grand canonical methods aid in the sampling of these water molecules, where conventional molecular dynamics equilibration times are often long, by allowing waters to be inserted and deleted from the system, according to the chemical potential. Here, we present our open source Python module, grand (https://github.com/essex-lab/grand), which allows molecular dynamics simulations to be performed in conjunction with grand canonical Monte Carlo sampling, using the OpenMM simulation engine. We demonstrate the accuracy of this module by reproducing the density of bulk water observed from constant pressure simulations. Application of this code to the bovine pancreatic trypsin inhibitor protein reproduces three buried crystallographic water sites that are poorly sampled using conventional molecular dynamics.

Conserved internal hydration motifs in protein kinases.

Crystal structures of diverse protein kinase catalytic subunits reveal a number of water molecules whose positions within the protein core are better preserved than amino acid types in many functionally important locations. It remains unknown whether they play any particular role, and whether their removal, disturbing local interaction patterns to no smaller degree than amino acid mutations, can affect kinase stability and function. In this study, we apply an array of computational approaches to characterize hydration of kinase catalytic subunits. First, we systematically screen multiple crystal structures with the use of a simplified hydration model in order to determine the distribution of internal solvent and the degree of its conservation. Second, we analyze water structure, dynamics and binding affinity to buried hydration sites in a number of kinases, also taking into account their variable functional state. We find that a large portion of buried solvent is dynamic, possibly contributing to kinase conformational changes related to the activation process. In turn, binding free energies of some of tightly bound conserved water molecules to different kinases tend to shift in a similar manner following the change of their functional state. This finding highlights the likely specific role of internal solvent in fine tuning local protein plasticity.

Application of pseudo-3D Chirp sub-bottom profiler survey: a case study of ancient wooden shipwreck site, west coast of Korea

Chirp sub-bottom profilers (SBP) provide centi-to-decimetre resolution, seismic data with applications for various geophysical and geological purposes. To verify the field application of imaging of a buried target with a cost-effective and easy-to-apply pseudo-3D Chirp SBP survey, we explored the buried site of an ancient wooden shipwreck off the west coast of Korea before underwater excavations. The survey was conducted using a commercial 2D Chirp SBP system with a newly devised recording system that preserved the true polarity of the chirp signal. To produce high-resolution 3D Chirp SBP data from 2D Chirp SBP datasets recorded by the novel system, an optimal data processing sequence, consisting of a first phase of 2D data processing and a second phase of 3D data processing was designed. The first, 2D phase, included the estimation of a source sweep signature, cross-correlation, and deconvolution using an inverse filter. The resulting resolution of the 2D Chirp SBP data was better than that of the enveloped data provided by the commercial acquisition system. The second phase of 3D data processing included gathering 3D datasets, redistributing of ping positions, and static correction. To improve the consistency of the seismic events and reduce the repetitive corrections (swell, tidal, tie, and residual corrections), a static correction was based on multi-beam echo sounder data. The amplitude variation near the shipwreck was clearly apparent in the time slice from the final pseudo-3D Chirp SBP dataset with a bin size of 2.0 m (crossline) × 0.6 m (inline). Through 3D rendering, the buried ancient shipwreck with dimensions of 5 m (width) × 12 m (length) × 2 m (depth) was imaged successfully.

Geophysical reconstruction of buried geological features and site effects estimation of the Middle Valle Umbra basin (central Italy)

The Middle Valle Umbra (central Italy) is a NW-SE 20 km long and 10 km wide Quaternary extensional basin located in the internal sector of the Apennine chain. This area historically experienced strong earthquakes that caused significant damages to the outstanding historical heritage. The same area has been recently hit by the 2016 seismic sequence of Amatrice-Visso-Norcia.With the aim to reconstruct the buried geological structures of the basin, a multi-technique geophysical approach was performed. An extended campaign of ambient noise measurements was carried out to investigate the subsurface setting, and to identify the main geological units. We performed three 2D passive arrays to analyze two different sites within the basin; their aperture was between 150 and 752 m for one site and of 48 m for the other site, to characterize the geological units in terms of sediment thickness and shear-wave velocity profile. Data collected were processed with f-k and MSPAC analysis to extract dispersion curves with good resolution in a frequency range of 0.5–10 Hz and 4.5–18 Hz for the two sites respectively. Spectral ratios were computed for every single station ambient noise measurement performed and for all the stations of the bigger array. Our final target is to extend these results to the whole valley, in order to retrieve the attitude of the main geological units and propose a reliable reconstruction of the subsurface geometry of the basin. Another point of this work is to evaluate the site response in the middle of the valley through the analysis of the earthquakes recorded by the accelerometric station IT.CSA (belonging to the Italian Civil Protection) and the corresponding recordings of the nearby rock station IT.ASS.

The determination of depth anomaly in archaeo-magnetic using an Euler deconvolution: Case study in Kuta Lubok fortress

Geophysical surveys have been conducted using magnetic in the archaeological site of Kuta Lubok fortress, Aceh Besar. The fortress was built in 13th century by the Lamuri Sultanate in the northern part Aceh coast. To map an estimated area of archaeological objects, 420 stations have been measured. The data were measured by 21 profiles across of the area with the length of profiles vary from 40 to 60 m. To get a complete resolution, data is taken with a distance of 20 m between lines and a distance of 5 m between stations. From the total magnetic field map that has reduced to the poles, the anomaly is generally able to delineate buried building sites. Then based on Euler deconvolution analysis, the structure of the fortress building can be seen using a 5-meter depth level. But a clearer structure is seen in the use of 10 m in depth. Based on the interpretation of the two tolerance layers it can be concluded that the Kuta Lubok fortress was found stretching parallel to the line along the 400 m to the east of the fort building which is still intact.

Fast Screening of Inhibitor Binding/Unbinding Using Novel Software Tool CaverDock.

Protein tunnels and channels are attractive targets for drug design. Drug molecules that block the access of substrates or release of products can be efficient modulators of biological activity. Here, we demonstrate the applicability of a newly developed software tool CaverDock for screening databases of drugs against pharmacologically relevant targets. First, we evaluated the effect of rigid and flexible side chains on sets of substrates and inhibitors of seven different proteins. In order to assess the accuracy of our software, we compared the results obtained from CaverDock calculation with experimental data previously collected with heat shock protein 90α. Finally, we tested the virtual screening capabilities of CaverDock with a set of oncological and anti-inflammatory FDA-approved drugs with two molecular targets—cytochrome P450 17A1 and leukotriene A4 hydrolase/aminopeptidase. Calculation of rigid trajectories using four processors took on average 53 min per molecule with 90% successfully calculated cases. The screening identified functional tunnels based on the profile of potential energies of binding and unbinding trajectories. We concluded that CaverDock is a sufficiently fast, robust, and accurate tool for screening binding/unbinding processes of pharmacologically important targets with buried functional sites. The standalone version of CaverDock is available freely at https://loschmidt.chemi.muni.cz/caverdock/ and the web version at https://loschmidt.chemi.muni.cz/caverweb/.