G Protein-Coupled Receptors (GPCRs) mediate signal transduction across cellular membranes and are major drug targets. Activation of these receptors upon binding of an extracellular ligand involves propagation of structural change across the transmembrane domain to the cytoplasmic G protein partner, a process generally thought to involve dynamic hydrogen(H)-bond networks. Here we present DNET, a graph-based tool and workflow that enables efficient computations of dynamic protein-water H-bond networks. DNET is a fully portable Python tool that reads simulation trajectories, computes graphs of the dynamic protein-water H-bond networks, and generates, for each H-bonding residue, a residue summary that includes water interactions, H-bond time series, histograms, potential of mean force estimates, and the number of conformations of the H-bond. To facilitate estimates of pKa fluctuations within the H-bond network, DNET calls PROPKA and computes, for each titratable residue that is part of the H-bond network, time series and analyses of the pKa estimates. To illustrate the usefulness of DNET we apply it to study the wild-type and two mutations of jumping spider rhodopsin 1, JSR-1, a visual rhodopsin GPCR activated by the photoisomerization of the covalently bound retinal chromophore. The UV-vis data we present here demonstrate that the mutated JSR-1 proteins express, but both have an altered electrostatic environment of the retinal Schiff base. The DNET analyses indicate a highly complex dynamics of the retinal H-bond network, with some H-bonds that have only one conformational mode, and other H-bonds with multiple conformational modes separated by small energy barriers, and pKa fluctuations that associate with the H-bond dynamics. The mutations associate with an altered H-bond network of the retinal Schiff base.

Microbial pump rhodopsins are highly versatile light-driven membrane proteins that couple protein conformational dynamics with ion translocation across the cell membranes. Understanding how microbial pump rhodopsins use specific amino acid residues at key functional sites to control ion selectivity and ion pumping direction is of general interest for membrane transporters, and could guide site-directed mutagenesis for optogenetics applications. To enable direct comparisons between proteins with different sequences we implement, for the first time, a unique numbering scheme for the microbial pump rhodopsin residues, NS-mrho. We use NS-mrho to show that distinct microbial pump rhodopsins typically have hydrogen-bond networks that are less conserved than anticipated from the amino acid residue conservation, whereas their hydrophobic interaction networks are largely conserved. To illustrate the role of the hydrogen-bond networks as structural elements that determine the functionality of microbial pump rhodopsins, we performed experiments, atomic-level simulations, and hydrogen bond network analyses on GR, the outward proton pump from Gloeobacter violaceus, and KR2, the outward sodium pump from Krokinobacter eikastus. The experiments indicate that multiple mutations that recover KR2 amino acid residues in GR not only fail to convert it into a sodium pump, but completely inactivate GR by abolishing photoisomerization of the retinal chromophore. This observation could be attributed to the drastically altered hydrogen-bond interaction network identified with simulations and network analyses. Taken together, our findings suggest that functional specificity could be encoded in the collective hydrogen-bond network of microbial pump rhodopsins.

Changes in structure and dynamics elicited by agonist ligand binding at the extracellular side of G protein coupled receptors (GPCRs) must be relayed to the cytoplasmic G protein binding side of the receptors. To decipher the role of water-mediated hydrogen-bond networks in this relay mechanism, we have developed graph-based algorithms and analysis methodologies applicable to datasets of static structures of distinct GPCRs. For a reference dataset of static structures of bovine rhodopsin solved at the same resolution, we show that graph analyses capture the internal protein-water hydrogen-bond network. The extended analyses of static structures of rhodopsins and opioid receptors suggest a relay mechanism whereby inactive receptors have in place much of the internal core hydrogen-bond network required for long-distance relay of structural change, with extensive local H-bond clusters observed in structures solved at high resolution and with internal water molecules. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.

Some studies have used physical techniques for the assessment of bone structure and composition. However, very few studies applied multiple techniques, such as those described below, at the same time. The aim of our study was to determine the chemical and mineralogical/organic composition of bovine tibial bone samples using geophysical/geochemical reference techniques. X-ray diffraction (XRD), thermogravimetry (TG), Fourier-transform infrared spectrometry with attenuated total reflectance accessory (FTIR-ATR), inductively coupled mass spectrometry (ICP-MS) and inductively coupled optical emission spectrometry (ICP-OES) were applied to measure the organic and inorganic composition of 14 bovine bone samples. In addition, peripheral quantitative CT (QCT) was used to assess BMD in these bones. We were able to define the total composition of the studied bone samples. ICP-OES and ICP-MS techniques were used to determine the major and trace element composition. The X-ray analysis could detect inorganic crystalline compounds of bones, such as bioapatite, and its degree of ordering, indicating whether the bones belong to a younger or older individual. The total volatile content of the samples was calculated using TG and resulted in about 35 weight% (wt%). This, together with the 65 wt% total resulting from the chemical analysis (i.e., inorganic components), yielded a total approaching 100 wt%. As a large portion of the volatile content (H2O, CO2, etc.) was liberated from the organic components and, subordinately, from bioapatite, it could be concluded that the volatile-to-solid ratio of the examined bone samples was ~35:65. The FTIR-ATR analysis revealed that the organic portion consists of collagens containing amide groups, as their typical bands (OH, CH, CO, NC) were clearly identified in the infrared spectra. Numerous parameters of bone composition correlated with BMD as determined by QCT. In conclusion, we performed a complex evaluation of bovine bones to test multiple geophysical/geochemical techniques in bone research in association with QCT bone densitometry. From a medical point of view, the composition of the studied bones could be reliably examined by these methods.

pH-dependent reactions at membrane interfaces are used by cells to maintain pH homeostasis and to ensure normal cell function. Membrane-bound proteins and peptide systems that sense pH typically bind protons using a few titratable sidechains that communicate with the bulk. The identity of these pH-sensing sidechains is central to hypotheses about protein reaction mechanisms and for the rational design of proton detectors. But, as protein groups hypothesized to sense pH in proteins and peptide systems are part of dynamic water-mediated networks, evaluating their pH-sensing functionality may prove difficult. We developed efficient graph-based algorithms and methodologies to evaluate dynamic hydrogen-bond networks of protein and peptide systems known to have a pH-sensing functionality, and to identify sites of hydrogen-bond networks where protons are likely to bind. We find that the assembly of dynamic water-mediated hydrogen-bond networks that include potential proton-sensing groups is a key event along the reaction coordinates of pH-dependent membrane proteins and membrane-bound peptides, and implement graph-based algorithms to characterize the dynamics of these water networks. We further find that polar and basic protein sidechains largely govern the dynamics of the water-mediated hydrogen-bond networks of proton-binding groups, and help maintain acidic sidechains within clusters that collectively can bind and store protons. Taken together, the ensemble of hydrogen-bond network computations we performed for pH-coupled proteins and peptide systems provide a framework to decipher the roles of dynamic water-mediated hydrogen-bond networks in proton binding at membrane interfaces. Research was supported in part by the European Union's Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement No 860592, Innovative Training Network ‘Proton and proton-coupled transport’, and by computing time from the Physics Department of the Freie Universität Berlin and from the Forschungszentrum Jülich (JURECA-DC, allocation PHDPORES).

Kalium channelrhodopsin 1 from Hyphochytrium catenoides ( Hc KCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K + over Na + in the absence of the canonical tetrameric K + selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin ( Hc CCR), a Na + channel with >100-fold larger Na + to K + permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K + versus Na + selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating.

Microbial rhodopsins are membrane proteins that use the energy absorbed by the covalently bound retinal chromophore to initiate reaction cycles resulting in ion transport or signal transduction. Thousands of distinct microbial rhodopsins are known and, for many rhodopsins, three-dimensional structures have been solved with structural biology, including as entire sets of structures solved with serial femtosecond crystallography. This sets the stage for comprehensive studies of large datasets of static protein structures to dissect structural elements that provide functional specificity to the various microbial rhodopsins. A challenge, however, is how to analyze efficiently intra-molecular interactions based on large datasets of static protein structures. Our perspective discusses the usefulness of graph-based approaches to dissect structural movies of microbial rhodopsins solved with time-resolved crystallography.

Dynamic hydrogen bonds and hydrogen bond networks give proteins structural plasticity required for function. This includes long-distance conformational coupling between remote regions of the protein, and transfer of protons across long distances by membrane transporters. In the case of large proteins and macromolecular complexes, identifying hydrogen-bond networks that assemble transiently in fluid environments brings about the challenge of large numbers of interactions that need to be evaluated.

Opioid receptors are G-protein-coupled receptors (GPCRs) part of cell signaling paths of direct interest to treat pain. Pain may associate with inflamed tissue characterized by acidic pH. The potentially low pH at tissue targeted by opioid drugs in pain management could impact drug binding to the opioid receptor, because opioid drugs typically have a protonated amino group that contributes to receptor binding, and the functioning of GPCRs may involve protonation change. In this review, we discuss the relationship between structure, function, and dynamics of opioid receptors from the perspective of the usefulness of computational studies to evaluate protonation-coupled opioid-receptor interactions.

Dynamic hydrogen-bond networks provide proteins with structural plasticity required to translate signals such as ligand binding into a cellular response or to transport ions and larger solutes across membranes and, thus, are of central interest to understand protein reaction mechanisms. Here, we present C-Graphs, an efficient tool with graphical user interface that analyzes data sets of static protein structures or of independent numerical simulations to identify conserved, vs unique, hydrogen bonds and hydrogen-bond networks. For static structures, which may belong to the same protein or to proteins with different sequences, C-Graphs uses a clustering algorithm to identify sites of the hydrogen-bond network where waters are conserved among the structures. Using C-Graphs, we identify an internal protein-water hydrogen-bond network common to static structures of visual rhodopsins and adenosine A2A G protein-coupled receptors (GPCRs). Molecular dynamics simulations of a visual rhodopsin indicate that the conserved hydrogen-bond network from static structure can recruit dynamic hydrogen bonds and extend throughout most of the receptor. We release with this work the code for C-Graphs and its graphical user interface.

Corona virus spike protein S is a large homo-trimeric protein anchored in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, and contribute symmetrically to a relatively large H-bond cluster at the receptor binding domain, and to a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.

Protein and protein-water hydrogen bonds shape the conformational energy landscape of G Protein-Coupled Receptors, GPCRs. As numerous static structures of GPCRs have been solved, the important question arises whether GPCR structures and GPCR conformational dynamics could be described in terms of conserved hydrogen-bond networks, and alterations of these hydrogen-bond networks along the reaction coordinate of the GPCR. To enable efficient analyses of the hydrogen-bond networks of GPCRs we implemented graph-based algorithms, and applied these algorithms to static GPCR structures from structural biology, and from molecular dynamics simulations of two opioid receptors. We find that static GPCR structures tend to have a conserved, core hydrogen-bond network which, when protein and water dynamics are included with simulations, extends to comprise most of the interior of an inactive receptor. In an active receptor, the dynamic protein-water hydrogen-bond network spans the entire receptor, bridging all functional motifs. Such an extensive, dynamic hydrogen-bond network might contribute to the activation mechanism of the GPCR.

In this research we propose to understand how opioid drugs used in the treatment of severe pain bind to membrane-embedded receptors in cells that are found within the low pH environment characteristic for injured and inflamed tissue. Opioid compounds are most potent known painkillers [1]. However, synthetic opioids, such as fentanyl can also be highly additive. In the past few years opioid addiction has become a problem of great concern due to the fact that in the United States the number of deaths that were the consequence of synthetic opioid overdose has risen dramatically (the USA opioid crisis). Therefore, it is of significant interest to understand how opioid compounds bind and active opioid receptors and how this knowledge can be used in the design of novel and safer painkillers. The receptors to which opioid drugs bind are classified into three distinct subtypes, which are the κ, δ and μ subtype. Most of the painkilling effects, as well as unwanted side effects stem from the activation of the μ opioid receptor, however recent research is emphasizing the importance of the other two as well. To date, most strategies in drug development of novel opioids have focused on compounds that activate opioid receptors both in the central nervous system and at the peripheral parts of the body. Such compounds are not tissue-selective, as they activate opioid receptors in both healthy and diseased tissue. The indiscriminate binding leads to side effects typical for opioids, which are the consequence of them binding to normal brain tissue. However a number of pathologies associated with severe pain, such as neuropathy, can be treated by acting specifically on peripheral neurons [2]. These pathologies are accompanied by inflammation, which causes local acidosis (low pH) of the diseased tissue. This enables tissue-specific drug targeting, whereby drugs would be active only at low pH, thus circumventing many side effects caused by opioids binding to receptors located elsewhere. Our collaborator Prof. Christoph Stein has developed a fluorinated fentanly analogue that is activated only in inflamed tissue with pH below 7 [3]. All opioid drugs have a protonated amine group; by attaching a fluorine atom to the fentanyl molecule, the pKa of the amine group is reduced, and the fluorinated fentanyl is protonated only at the low pH characteristic to inflamed tissue. The protonated fluorinated fentanyl can then bind to the opioid receptor by salt bridging to an aspartate group of the receptor. Low pH of the tissue leads to changes in the protonation states within the receptor. However, protonation-coupled dynamics of opioid receptors and their interactions with drugs is poorly understood. We know that changes in the protonation state of internal protein groups can impact significantly the local protein structure and dynamics [4]. As a first step towards understanding the pHdependent binding of opioids to opioid receptors, we plan to perform molecular dynamic simulations to study the binding of opioid drugs to membraneembedded receptors with different protonation states of the receptor and of the drug molecule (Fig. 1).

The past decade has seen a great number of studies dealing with magmatic water contents and how these could be retrieved by the nominally anhydrous minerals’ (NAMs) trace structural hydroxyl (water) contents. Constraints have been made to magmatic hygrometry with clinopyroxene and plagioclase. Although results suggest that the method is more flexible and reliable than melt inclusion studies, they also indicate that the trace hydroxyl contents could still be overprinted by syn- and post-eruptive processes. Clinopyroxenes can hold more structural hydroxyl than plagioclases. A comprehensive review is presented with the inclusion of all published results so far to compile the available pieces of information. As a case study, micro-FTIR measurements are made of a representative set of plagioclase phenocrysts from the Börzsöny Mts. (Carpathian–Pannonian Region). The samples were selected to represent the progress of the volcanic activity in time and space, considering the petrologic and geochemical evolution of volcanic products in well-defined volcanostratigraphic positions. The syn- and post-eruptive cooling rate seems to have the greatest effect on water retention. This means that the systematic investigation of water in volcanic phenocrysts can contribute to distinguish the slowly and rapidly cooling parts of the volcanostratigraphic units.

Geochemical characteristics of sediments are responses to physical and chemical alteration in landslides. However, consequences of in situ interactions associated with landslides are difficult to distinguish from those related to long-term weathering in young soft sediments such as loess. In this study, geochemical characteristics of the Kulcs landslide in Hungary are studied to identify the provenance of the loess–paleosol–red clay sequence and geochemical signatures that can potentially be attributed to the effects of landsliding. Results indicate that sliding is largely initiated by the lithological changes within the landslide body. Sediments above the sliding zone closely resemble the non-slipped Pleistocene old loess deposits from Hungary. It is also confirmed that the sliding zone develops in old paleosols in the loess sequence and red clays at its base which are all characterized by the enrichment of Al, K, Na, H2O and considerable depletion in Ca and Mg associated with carbonates. Altogether, these geochemical characteristics indicate that chemical weathering trend of unconsolidated landslide sediments is slightly modified by the redistribution of carbonates and decomposition of plagioclase. It is assumed that the distribution of Mn and Ba is modified by the water–sediment interaction in the landslide.

The structural similarities between the inorganic component of bone tissue and geological formations make it possible that mathematic models may be used to determine weight percentage composition of different mineral element oxides constituting the inorganic component of bone tissue. The determined weight percentage composition can be verified with the determination of element oxide concentration values by laser induced plasma spectroscopy and inductively coupled plasma optical emission spectrometry. It can be concluded from calculated weight percentage composition of the inorganic component of bone tissue and laboratory analyses that the properties of bone tissue are determined primarily by hydroxylapatite. The inorganic bone structure can be studied well by determining the calcium oxide concentration distribution using the laser induced plasma spectroscopy technique. In the present study, thin polished bone slides prepared from male bovine tibia were examined with laser induced plasma spectroscopy in a regular network and combined sampling system to derive the calculated calcium oxide concentration distribution. The superficial calcium oxide concentration distribution, as supported by "frequency distribution" curves, can be categorized into a number of groups. This, as such, helps in clearly demarcating the cortical and trabecular bone structures. Following analyses of bovine tibial bone, the authors found a positive association between the attenuation value, as determined by quantitative computer tomography and the "ρ" density, as used in geology. Furthermore, the calculated "ρ" density and the measured average calcium oxide concentration values showed inverse correlation.

The effect of the carrier gas flow rate on laser ablation inductively coupled mass spectrometry (LA-ICP-MS) signals of brass components (Cu, Zn, and Ni) were studied primarily with the use of a conventional closed ablation cell (UP-213) and with the use of an open ablation cell of our own construction. In the closed ablation cell configuration with the carrier gas flow rate in the range of 0.40–1.3 L min− 1 Ar, the MS signals increased significantly (an 8.2-fold increase for a Cu signal), and the Zn/Cu signal ratio increased 3.2 times. To identify the degree of fractionation, the conventional solution sample introduction method was selected as the reference method because it is expected free from fractionation for Cu and Zn. To obtain a theoretical value, calculations were made based on Saha's relationship of ionization, which resulted in fair agreement with the experimental results of the solution method. By comparing the Zn/Cu signal ratios obtained from both the LA and the solution method, a fractionation factor of 2.26 was deduced for these two components. To explain the increased signals described above, the transport efficiency as a function of the carrier gas flow rate was calculated for different particle size fractions based on existing theories. It was demonstrated that the large increase in the signals with carrier gas flow rate is predominantly due to processes taking place in the ablation cell (i.e., neither during the transportation nor in the ICP). The results of the novel fundamental works on aerosol formation under LA conditions were considered and complemented with the application of the Kelvin (Gibbs-Thomson) equation to calculate critical sizes of aerosol particles in the nucleating vapors of Cu, Zn, Ni and Pb elements. It was concluded that the noted increase of signals was due to the intensification of mixing of the expanding vapor-cloud with cold gas when applying an increasing carrier gas flow rate. It was also concluded that the fractionation factor mentioned above for Zn/Cu components (2.26) was the consequence of fractional vaporization. The open ablation cell and aerosol transporting pump system did not satisfy the demands of the sensitive LA-ICP-MS determinations. The primary reason for this was the low efficiency of the miniature pump used for aerosol transportation.

The evapotranspiration zones connected to the local flow systems were studied in the Danube-Tisza interfluvial area of the Great Hungarian Plain in order to understand the origin of high arsenic concentrations in shallow groundwater. Monitoring of five wells at two sites was carried out for almost one year. In each sample of the five wells the arsenic concentration was above the 10 mu g/L drinking water standard, while the groundwater in one of the wells had almost 600 mu g/L arsenic. Field separation of arsenic species was done in order to preserve their oxidation states in the groundwater samples. At four of the wells 70-80% of the arsenic was present as As3+, while at one well only in 40%. Both oxidative and reductive geochemical environments were found. The water samples show the effects of evapotranspiration, but other factors, such as microbiological activity, probably promote the remobilization of arsenic into groundwater.