Ordered Water Molecules Research Articles

Solid state NMR spectral editing of histidine, arginine and lysine using Hadamard encoding.

The NMR signals from protein sidechains are rich in information about intra- and inter-molecular interactions, but their detection can be complicated due to spectral overlap as well as conformational and hydrogen exchange. In this work, we demonstrate a protocol for multi-dimensional solid-state NMR spectral editing of signals from basic sidechains based on Hadamard matrix encoding. The Hadamard method acquires multi-dimensional experiments in such a way that both the backbone and under-sampled sidechain signals can be decoded for unambiguous editing in the 15 N spectral frequency dimension. All multi-dimensional 15 N-edited solid-state NMR experiments can be acquired using this strategy, thereby accelerating the acquisition of spectra spanning broad frequency bandwidth. Application of these methods to the ferritin nanocage, reveals signals from N atoms from His, Arg, Lys and Trp sidechains, as well as their tightly bound, ordered water molecules. The Hadamard approach adds to the arsenal of spectroscopic approaches for protein NMR signal detection.

Probing the Electrostatic Effects of H-Ras Tyrosine 32 Mutations on Intrinsic GTP Hydrolysis Using Vibrational Stark Effect Spectroscopy of a Thiocyanate Probe.

The wildtype H-Ras protein functions as a molecular switch in a variety of cell signaling pathways, and mutations to key residues result in a constitutively active oncoprotein. However, there is some debate regarding the mechanism of the intrinsic GTPase activity of H-Ras. It has been hypothesized that ordered water molecules are coordinated at the active site by Q61, a highly transforming amino acid site, and Y32, a position that has not previously been investigated. Here, we examine the electrostatic contribution of the Y32 position to GTP hydrolysis by comparing the rate of GTP hydrolysis of Y32X mutants to the vibrational energy shift of each mutation measured by a nearby thiocyanate vibrational probe to estimate changes in the electrostatic environment caused by changes at the Y32 position. We further compared vibrational energy shifts for each mutation to the hydration potential of the respective side chain and demonstrated that Y32 is less critical for recruiting water molecules into the active site to promote hydrolysis than Q61. Our results show a clear interplay between a steric contribution from Y32 and an electrostatic contribution from Q61 that are both critical for intrinsic GTP hydrolysis.

Molecular basis for pH sensing in the KDEL trafficking receptor

Trafficking receptors control protein localization through the recognition of specific signal sequences that specify unique cellular locations. Differences in luminal pH are important for the vectorial trafficking of cargo receptors. The KDEL receptor is responsible for maintaining the integrity of the ER by retrieving luminally localized folding chaperones in a pH-dependent mechanism. Structural studies have revealed the end states of KDEL receptor activation and the mechanism of selective cargo binding. However, precisely how the KDEL receptor responds to changes in luminal pH remains unclear. To explain the mechanism of pH sensing, we combine analysis of X-ray crystal structures of the KDEL receptor at neutral and acidic pH with advanced computational methods and cell-based assays. We show a critical role for ordered water molecules that allows us to infer a direct connection between protonation in different cellular compartments and the consequent changes in the affinity of the receptor for cargo.

Thin Sputtered Titanium Nitride Layers for Corrosion Protection of 316L Stainless Steel Under PEM Water Electrolyzer Anode-like Conditions

Proton exchange membrane water electrolyzers (PEMWE) electrochemically split the water molecule in order to produce high-purity oxygen (at the anode) and hydrogen (at the cathode). When coupled to a renewable energy source (such as wind or sun), green hydrogen can be synthesized, which may contribute to a massive reduction in carbon dioxide emissions in the so-called “hard-to-abate” industrial sectors, like steel and cement manufacturing.Yet, the elevated capital expenditures (capex) deriving from the use of expensive corrosion-resistant materials undermine the economic competitiveness of the PEMWE technology compared to the main hydrogen production processes, like steam reforming. To date, on the PEMWE anode side, metallic bipolar plates (BPP) and porous transport layers (PTL), are essentially made of titanium coated with precious metal layers (platinum, gold) so as to withstand the corrosive and oxidizing environment. Consequently, BPP and PTL account for more than 60% of the capex of the PEMWE stack [1,2]. Without corrosion protection, the harsh operating anodic conditions may lead to performance-killing release of cationic elements from the BPP and PTL into the catalyst layer and the polymer electrolyte [3]. It may also cause the growth of resistive oxide layers at the BPP and PTL surfaces and the development of undesired interfacial contact resistance (ICR).This contribution will focus on the corrosion protection of the anode side of BPP and will first briefly review the most recent investigations driven by capex reduction, notably based on the replacement of titanium by stainless steels and the application of novel, more cost-effective anti-corrosion coatings [3].In a second part, results on the corrosion protection of 316L stainless steel by thin layers of titanium nitride prepared by DC magnetron sputtering will be presented. The thin TiN coatings were deposited on glass, silicon and 316L stainless steel substrates using a titanium target reacting with nitrogen gas. The crystalline phase, the nanostructure and the composition of the thin films were characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray reflectivity, energy dispersive X-ray spectroscopy and electron energy loss spectroscopy. The corrosion resistance of the TiN-coated stainless steel was evaluated ex situ using open-circuit potential monitoring and electrochemical impedance spectroscopy in dilute H2SO4 solution in a three-electrode electrochemical cell. The interfacial contact resistance was assessed before and after corrosion testing. The protective properties of titanium nitride coatings on 316L under PEMWE anode-like conditions will be discussed.Acknowledgements The authors gratefully acknowledge the financial support of the French National Research Agency (agreement ANR-22-PEHY-0009 under the France 2030 program as well as agreement ANR-22-CE93-0008-01).

Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase

Cytochrome c oxidase (C c O) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a 3 of ba 3 -type C c O. In contrast with the aa 3 -type enzyme, our data show how CO is stabilized on Cu B through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the Cu B -CO complex in ba 3 -type C c O and, by extension, the extremely high oxygen affinity of the enzyme.

Anion-Specific Adsorption of Carboxymethyl Cellulose on Cellulose.

Integration of fiber modification step with a modern pulp mill is a resource efficient way to produce functional fibers. Motivated by the need to integrate polymer adsorption with the current pulping system, anion-specific effects in carboxymethylcellulose (CMC) adsorption have been studied. The QCM-D adsorption experiments revealed that CMC adsorption to the cellulose model surface is prone to anion-specific effects. A correlation was observed between the adsorbed CMC and the degree of hydration of the co-ions present in the magnesium salts. The presence of a chaotropic co-ion such as nitrate increased the adsorption of CMC on cellulose compared to the presence of the kosmotropic sulfate co-ion. However, anion-specificity was not significant in the case of salts containing zinc cations. The hydration of anions determines the distribution of the ions at the interface. Chaotropic ions, such as nitrates, are likely to be distributed near the chaotropic cellulose surface, causing changes in the ordering of water molecules and resulting in greater entropy gain once released from the surface, thus increasing CMC adsorption.

Modeling the influence of the external electric fields on water viscosity inside carbon nanotubes.

Equilibrium molecular dynamics simulations were performed to explore the effects of external electric fields and confinement on water properties inside various carbon nanotubes (CNTs). Using different GHz electric field frequencies as well as various constant electric field strengths, the radial distribution function and density profile were investigated, by which the impact of the electric fields and confinement on the water structure are revealed. The results indicated water molecules inside the CNT form layered structures due to topological confinement applying external electric fields can disturb this ordered water molecules structure and increase the viscosity of confined water, particularly in the case of CNTs with a radius less than 13.5Å. Conversely, for CNTs with a radius greater than13.5Å, the viscosity decreases under the influence of external oscillating or constant electric fields. How dose the synergism of confinement and external electric fields affect the water properties inside the CNTs?

Turning an energy-based defect detector into a multi-molecule structural indicator for water.

Recent studies have provided conclusive evidence for the existence of a liquid-liquid critical point in numerical models of water. Such a scenario implies the competition between two local molecular arrangements of different densities: a high-density liquid (HDL) and a low-density liquid (LDL). Within this context, the development of accurate structural indicators to properly characterize the two interconverting local structures is demanded. In a previous study, we introduced a reliable energy-based structural descriptor that properly discriminates water molecules into tetrahedrally arranged molecules (T molecules) and distorted molecules (D molecules). The latter constitute defects in terms of hydrogen bond (HB) coordination and have been shown to represent a minority component, even at high temperatures above the melting point. In addition, the D molecules tend to form high-quality HBs with three T molecules and to be surrounded by T and D molecules at further distances. Thus, it became evident that, while the LDL state might consist of a virtually pure T state, the HDL state would comprise mixed molecular arrangements including the D molecules. Such a need to abandon the single-molecule description requires the investigation of the degree of structural information to be incorporated in order to build an appropriate multi-molecule indicator. Hence, in this work, we shall study the effect of the local structural constraints on the water molecules in order to discriminate the different molecular arrangements into two disjoint classes. This will enable us to build a multi-molecule structural indicator for water whose performance will then be investigated within the water's supercooled regime.

Biophysical Properties of Lipid Membranes from Barley Roots during Low-Temperature Exposure and Recovery.

Glycerolipid remodeling, a dynamic mechanism for plant subsistence under cold stress, has been posited to affect the biophysical properties of cell membranes. In barley roots, remodeling has been observed to take place upon exposure to chilling stress and to be partially reverted during stress relief. In this study, we explored the biophysical characteristics of membranes formed with lipids extracted from barley roots subjected to chilling stress, or during a subsequent short- or long-term recovery. Our aim was to determine to what extent barley roots were able to offset the adverse effects of temperature on their cell membranes. For this purpose, we analyzed the response of the probe Laurdan inserted in bilayers of different extracts, the zeta potential of liposomes, and the behavior of Langmuir monolayers upon compression. We found important changes in the order of water molecules, which is in agreement with the changes in the unsaturation index of lipids due to remodeling. Regarding Langmuir monolayers, we found that films from all the extracts showed a reorganization at a surface pressure that depends on temperature. This reorganization occurred with an increase in entropy for extracts from control plants and without entropy changes for extracts from acclimated plants. In summary, some membrane properties were recovered after the stress, while others were not, suggesting that the membrane biophysical properties play a role in the mechanism of plant acclimation to chilling. These findings contribute to our understanding of the impact of lipid remodeling on biophysical modifications in plant roots.

Structural conservation of antibiotic interaction with ribosomes.

The ribosome is a major target for clinically used antibiotics, but multidrug resistant pathogenic bacteria are making our current arsenal of antimicrobials obsolete. Here we present cryo-electron-microscopy structures of 17 distinct compounds from six different antibiotic classes bound to the bacterial ribosome at resolutions ranging from 1.6 to 2.2 Å. The improved resolution enables a precise description of antibiotic-ribosome interactions, encompassing solvent networks that mediate multiple additional interactions between the drugs and their target. Our results reveal a high structural conservation in the binding mode between antibiotics with the same scaffold, including ordered water molecules. Water molecules are visualized within the antibiotic binding sites that are preordered, become ordered in the presence of the drug and that are physically displaced on drug binding. Insight into RNA-ligand interactions will facilitate development of new antimicrobial agents, as well as other RNA-targeting therapies.

Fe(III) Docking-Activated Sites in Layered Birnessite for Efficient Water Oxidation

Non-noble metal catalysts for promoting the sluggish kinetics of oxygen evolution reaction (OER) are essential to efficient water splitting for sustainable hydrogen production. Birnessite has a local atomic structure similar to that of an oxygen-evolving complex in photosystem II, while the catalytic activity of birnessite is far from satisfactory. Herein, we report a novel Fe-Birnessite (Fe-Bir) catalyst obtained by controlled Fe(III) intercalation- and docking-induced layer reconstruction. The reconstruction dramatically lowers the OER overpotential to 240 mV at 10 mA/cm2 and the Tafel slope to 33 mV/dec, making Fe-Bir the best of all the reported Bir-based catalysts, even on par with the best transition-metal-based OER catalysts. Experimental characterizations and molecular dynamics simulations elucidate that the catalyst features active Fe(III)-O-Mn(III) centers interfaced with ordered water molecules between neighboring layers, which lower reorganization energy and accelerate electron transfer. DFT calculations and kinetic measurements show non-concerted PCET steps conforming to a new OER mechanism, wherein the neighboring Fe(III) and Mn(III) synergistically co-adsorb OH* and O* intermediates with a substantially reduced O-O coupling activation energy. This work highlights the importance of elaborately engineering the confined interlayer environment of birnessite and more generally, layered materials, for efficient energy conversion catalysis.

Uncovering water effects in protein-ligand recognition: importance in the second hydration shell and binding kinetics.

Developing a ligand with high affinity for a specific protein target is essential for drug design, and water molecules are well known to play a key role in protein-drug recognition. However, predicting the role of particularly ordered water molecules in drug binding remains challenging. Furthermore, hydration free energy contributed from the water network, including the second shell of water molecules, is far from being well studied. In this research we focused on these aspects to accurately and efficiently evaluate water effects in protein-ligand binding affinity. We developed a new strategy using a free-energy calculation method, VM2. We successfully predicted the stable ordered water molecules in a number of protein systems: PDE 10a, HSP90, tryptophan synthase (TRPS), CDK2 and Factor Xa. In some of these, the second shell of water molecules appeared to be critical in protein-ligand binding. We also applied the strategy to largely improve binding free energy calculation using the MM/PBSA method. When applying MM/PBSA alone for two systems, CDK2 and Factor Xa, the computed binding free energy resulted in poor to moderate R2 values with experimental data. However, including water free energy correction greatly improved the free energy calculation. Furthermore, our work helped to explain how xk263 is a 1000 times faster binder to HIVp than ritonavir, a potentially useful tool for investigating binding kinetics. Our studies reveal the importance of fully considering water effects in therapeutic developments in pharmaceutical and biotechnology industries and for fundamental research in protein-ligand recognition.

A diminished hydrophobic effect inside the GroEL/ES cavity contributes to protein substrate destabilization

Confining compartments are ubiquitous in biology, but there have been few experimental studies on the thermodynamics of protein folding in such environments. Recently, we reported that the stability of a model protein substrate in the GroEL/ES chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution, but the origin of this effect remained unclear. Here, we show that this destabilization is caused, at least in part, by a diminished hydrophobic effect in the GroEL/ES cavity. This reduced hydrophobic effect is probably caused by water ordering due to the small number of hydration shells between the cavity and protein substrate surfaces. Hence, encapsulated protein substrates can undergo a process similar to cold denaturation in which unfolding is promoted by ordered water molecules. Our findings are likely to be relevant to encapsulated substrates in chaperonin systems, in general, and are consistent with the iterative annealing mechanism of action proposed for GroEL/ES.

An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

Structural basis for the transformation of the traditional medicine berberine by bacterial nitroreductase.

The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than EcNfsA, is responsible for the conversion of BBR to dhBBR in spite of a low reaction rate. The crystal structure of the EcNfsB-BBR complex showed that BBR binds into the active pocket at the dimer interface, and its large conjugated plane stacks above the plane of the FMN cofactor in a nearly parallel orientation. BBR is mainly stabilized by π-stacking interactions with both neighboring aromatic residues and FMN. Structure-based mutagenesis studies further revealed that the highly conserved Phe70 and Phe199 are important residues for the conversion of BBR. The structure revealed that the C6 atom of BBR (which receives the hydride) is ∼7.5 Å from the N5 atom of FMN (which donates the hydride), which is too distant for hydride transfer. Notably, several well ordered water molecules make hydrogen-bond/van der Waals contacts with the N1 atom of BBR in the active site, which probably donate protons in conjunction with electron transfer from FMN. The structure-function studies revealed the mechanism for the recognition and binding of BBR by bacterial NRs and may help to understand the conversion of BBR by the gut microbiota.

The structure of Deinococcus radiodurans transcriptional regulator HucR retold with the urate bound

HucR is a MarR family protein of Deinococcus radiodurans, which binds tightly to the intergenic region of HucR and the uricase gene to inhibit their expression. Urate (or uric acid) antagonizes the repressor function of HucR by binding to HucR to impede its association with the cognate DNA. The previously reported crystal structure of HucR was without the bound urate showing significant structural homology to other MarR structures. In this paper, we report the crystal structure of HucR determined with the urate bound. However, despite the fact that the urate is found at a site well-known to harbor ligands in other MarR family proteins, the overall HucR structure indicates that no significant change in structure takes place with the urate bound. Structure analysis further suggests that the urate interaction in HucR is mediated by histidine/glutamate side chains and ordered water molecules stabilized by various residues. Such interaction is quite unique compared to other known structural interactions between urate and its binding proteins. Furthermore, structural comparison of the apo- and the urate bound forms allows us to hypothesize that the Trp20-mediated water network in the apo-form stabilizes the proper HucR fold for cognate DNA binding, and that urate binding, also via Trp20, and the consequent reorganization of water molecules in the binding pocket, likely disrupts the DNA binding configuration to result in the attenuated DNA binding.

Computational hints for the simultaneous spectroscopic detection of common contaminants in water

In this work, we present a reliable computational methodology with the capability to aid in the identification of ionic water contaminants, such as nitrite, nitrate, and thiocyanate ions, based on the use of Resonance Raman (RR) spectroscopy. The method combines an exhaustive configurational sampling that fully captures the structural complexity inherent to aqueous solutions with state–of–the–art computational techniques that accurately simulate the response properties originating Resonance Raman signals of molecules in solution. Our computational findings show that it is possible to limit the quantum mechanical treatment to only a few explicit water molecules in order to capture the relevant interactions, and thus reproduce the available experimental spectra for NO2- and NO3-. Once validated, the methodology is applied to the prediction of the RR spectrum of aqueous SCN−. Our results indicate that by using an incident wavelength of 210 nm, the three emerging contaminants can be simultaneously detected in an aqueous matrix, thus avoiding the laborious indirect measurements used in current protocols. The designed protocol offers generalized explicit benefits: simultaneous detection of pollutants whose absorption spectra overlap, and because of the very nature of RR, pushes detection limits to lower concentrations.

Structured Water Molecules on Membrane Proteins Resolved by Atomic Force Microscopy.

Water structuring on the outer surface of protein molecules called the hydration shell is essential as well as the internal water structures for higher-order structuring of protein molecules and their biological activities in vivo. We now show the molecular-scale hydration structure measurements of native purple membrane patches composed of proton pump proteins by a noninvasive three-dimensional force mapping technique based on frequency modulation atomic force microscopy. We successfully resolved the ordered water molecules localized near the proton uptake channels on the cytoplasmic side of the individual bacteriorhodopsin proteins in the purple membrane. We demonstrate that the three-dimensional force mapping can be widely applicable for molecular-scale investigations of the solid-liquid interfaces of various soft nanomaterials.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Structural and molecular dynamics of ammonia transport in Staphylococcus aureus NH3-dependent NAD synthetase

Ammonia dependent NAD+ synthetase from multi drug resistance Staphylococcus aureus catalyzes ATP dependent formation of NAD+ from deamido-NAD+ and ammonia at the synthetase active site. Binding of ATP accompanies a large movement of flexible loop region (205-225) acting as a lid to the catalytic core. A 17 Å long ammonia tunnel with an entry and exit radius of 3.5 Å and 3.2 Å respectively allows transfer of ammonia from surface to the active site of the enzyme in each monomer to attack the C7N=O7N linkage of transient intermediate NAD-adenylate thus releasing NAD+. In this study, we report structural details of ammonia transport tunnel in Staphylococcus aureus NH3-dependent NAD synthetase and compared their architecture and dynamics with other bacterial and eukaryotic enzymes. Tunnel shows conformational variations in apo and substrate complexes and is less intricate compared to glutamine dependent counterparts. We have also performed steered molecular dynamic simulations of ammonia transport across the tunnel in enzyme-intermediate complex which reveals critical bottleneck residues and structural determinants during ammonium migration. Ordered water molecules and conserved charged residues form a network of hydrogen bonds and electrostatic interaction which facilitate the ammonium movement towards the active center. Analysis of the sMD simulated structural snapshots delineates the conformational reshaping of ammonia tunnel at the different step of the enzymatic reaction. Tunnel architecture and environment could offer the new target site to design novel small molecule inhibitors for the development of more efficient therapeutics against multi drug resistant S. aureus strains.