Internal Water Molecules Research Articles

Spectroscopic Studies on Maillard Reactions of Peanut Ara h 2.01 With Respective Reducing Sugars and Their Impacts on Ara h 2.01 Allergenicity.

The effects of the Maillard reactions of peanut Ara h 2.01 with respective glucose and xylose on its conformation and allergenicity were investigated. Circular dichroism (CD) spectral studies showed that short-term heating alone with reducing sugars did not change protein secondary structures, but after undergoing Maillard reactions, its secondary structures changed with some α-helices being transformed into β-sheets and random coils. Fluorescence spectral studies indicated that as temperature increased, protein became loose and extended induced by the reducing sugars. Long heat treatment at higher temperature caused protein collapse with the extrusion of its internal water molecules, which was hindered partially by the reducing sugars that entered the protein in Maillard reaction. Resonance light scattering (RLS) spectra confirmed that more xylose molecules entered protein due to its stronger interaction with the protein after Maillard reactions. ELISA assays exhibited that heat treatment at 100°C led to some allergenic epitopes being embedded inside protein molecules due to protein collapse and that partial epitopes were covered by the reducing sugars after Maillard reaction, resulting in decreases in its allergenicity. Also, xylose reduced the allergenicity of protein more efficiently owing to the stronger interaction between them. All above experimental results were explained reasonably by bioinformatic analysis and molecular docking.

Wet Conformation of Prion-Like Domain and Intimate Correlation of Hydration and Conformational Fluctuations.

Proteins with prion-like domains (PLDs) are involved in neurodegeneration-associated aggregation and are prevalent in liquid-like membrane-less organelles. These PLDs contain amyloidogenic stretches but can maintain dynamic disordered conformations, even in the condensed phase. However, the molecular mechanism underlying such intricate conformational properties of PLDs remains elusive. Here we employed molecular dynamics simulations to investigate the conformational properties of a prototypical PLD system (i.e., FUS PLD). According to our simulation results, PLD adopts a wet collapsed conformation, wherein most residues maintain sufficient hydration with the abundance of internal water. These internal water molecules can rapidly exchange between the protein interior and the bulk, enabling intensive coupling of the entire protein with its hydration environment. The dynamic exchange of water molecules is intimately correlated to the overall conformational fluctuations of PLD. Furthermore, the abundance of dynamic internal water suppresses the formation of aggregation-prone ordered structures. These results collectively elucidate the crucial role of internal water in sustaining the dynamic disordered conformation of the PLD and inhibiting its aggregation propensity.

Unique hydrogen-bonding network in a viral channelrhodopsin

Channelrhodopsins (CRs) are used as key tools in optogenetics, and novel CRs, either found from nature or engineered by mutation, have greatly contributed to the development of optogenetics. Recently CRs were discovered from viruses, and crystal structure of a viral CR, OLPVR1, reported a very similar water-containing hydrogen-bonding network near the retinal Schiff base to that of a light-driven proton-pump bacteriorhodopsin (BR). In both OLPVR1 and BR, nearly planar pentagonal cluster structures are comprised of five oxygen atoms, three oxygens from water molecules and two oxygens from the Schiff base counterions. The planar pentagonal cluster stabilizes a quadrupole, two positive charges at the Schiff base and an arginine, and two negative charges at the counterions, and thus plays important roles in light-gated channel function of OLPVR1 and light-driven proton pump function of BR. Despite similar pentagonal cluster structures, present FTIR analysis revealed different hydrogen-bonding networks between OLPVR1 and BR. The hydrogen bond between the protonated Schiff base and a water is stronger in OLPVR1 than in BR, and internal water molecules donate hydrogen bonds much weaker in OLPVR1 than in BR. In OLPVR1, the bridged water molecule between the Schiff base and counterions forms hydrogen bonds to D76 and D200 equally, while the hydrogen-bonding interaction is much stronger to D85 than to D212 in BR. The present interpretation is supported by the mutation results, where D76 and D200 equally work as the Schiff base counterions in OLPVR1, but D85 is the primary counterion in BR. This work reports highly sensitive hydrogen-bonding network in the Schiff base region, which would be closely related to each function through light-induced alterations of the network.

Graph-based algorithms to dissect long-distance water-mediated H-bond networks for conformational couplings in GPCRs.

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.

Structure Damage on Cotton Fiber via Coupling Effect of Moisture Regains and Low Temperature

ABSTRACT The most crucial variables that influence the characteristics and quality of cotton fibers during the cotton growth and ginning processes are temperature and moisture regains. Here the cotton fiber samples were generated with pre-treatment using a synergistic strategy of moisture regains and low temperatures, the morphology and structure of the fibers with different pre-treatments were analyzed by using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray Photoelectron Spectrometer (XPS), X-ray diffraction (XRD), and Carbon-13 Nuclear Magnetic Resonance Spectrometry (13C NMR). Further, the mechanical properties of the cotton fibers were evaluated using LLY-06E tensile. The results shown that, with 3 days of freezing in -18℃, the linear density variation of the cotton fiber samples reached maximum, and the surface of the fibers showed noticeable fractures, local holes, and folded damage. Moreover, the low temperature and high moisture regain caused the internal water molecules of the cotton fibers interact in complex ways, altering the intermolecular forces but without changing the chemical composition. This research expands the temperature field of cotton fibers, particularly in the ginning process, by investigating the damage to the mechanical characteristics of cotton fibers caused by the combined effect of low temperature and moisture regain.

Micro-destructive assessment of subgrade compaction quality using ultrasonic pulse velocity

The ultrasonic pulse velocity (UPV) correlates significantly with the density and pore size of subgrade filling materials. This research conducts numerous Proctor and UPV tests to examine how moisture and rock content affect compaction quality. The study measures the changes in UPV across dry density and compaction characteristics. The compacted specimens exhibit distinct microstructures and mechanical properties along the dry and wet sides of the compaction curve, primarily influenced by internal water molecules. The maximum dry density exhibits a positive correlation with the rock content, while the optimal moisture content demonstrates an inverse relationship. As the rock content increases, the relative error of UPV measurement rises. The UPV follows a hump-shaped pattern with the initial moisture content. Three intelligent models are established to forecast dry density. The measure of UPV and PSO-BP-NN model quickly assesses compaction quality.

Controllable Synthesis of Defective UiO-66 for Efficient Degradation and Detection of Ozone.

Metal-organic framework (MOF) structures have gained significant attention for their exceptional catalytic performance in ozone degradation, even under high humidity conditions, which is attributed to the presence of unsaturated metal sites (MOF defects). However, the correlation between MOF defects and catalytic ozone remains ambiguous, and a general approach for the controllable synthesis of high-performance MOF structures is currently lacking. Herein, different defective UiO-66 materials with cluster or ligand defects were obtained by precisely controlling small molecular acid modulators. Their catalytic performance can be analyzed in real time through the specific cataluminescence (CTL) signal of ozone at the interface. The presence of ligand defects was found to be crucial for both catalytic degradation and luminescence of ozone, and the CTL signal exhibited a positive correlation with the endogenous hydroxyl group content in the material (R2 = 0.982), while external humidity further supplemented internal water molecules within the material. Furthermore, theoretical calculations were conducted to compare the adsorption behaviors of ozone on the defective UiO-66 under dry/wet conditions, leading to the proposal of two potential reaction pathways. Subsequently, UiO-66-DA with superior catalytic performance was employed to develop a highly efficient CTL sensor capable of accurately detecting ozone (LOD = 23.3 ppb). This study held significant value in elucidating the reaction site of ozone on MOFs and achieving optimal catalytic effects through the careful selection of modulators and humidity levels.

Interfacial water molecules contribute to antibody binding to the receptor-binding domain of SARS-CoV-2 spike protein

Antibodies that recognize the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially the neutralizing antibodies, carry great hope in the treatment and final elimination of COVID-19. Driven by a synchronized global effort, thousands of antibodies against the spike protein have been identified during the past two years, with the structural information available at atomistic detail for hundreds of these antibodies. We developed an improved molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method including explicitly treated interfacial water to calculate the binding free energy between representative antibodies and the receptor binding domain (RBD) domain of SARS-COV-2 spike proteins. We discovered that explicit treatment of water molecules located at the interface between RBD and antibody effectively improves the results for the WT and variants of concern (VOC) systems. Interfacial water molecules, together with surface and internal water molecules, behave drastically from bulk water and exert peculiar impacts on protein dynamics and energy, and thus warrant explicit treatment to complement implicit solvent models. Our results illustrate the importance of including interfacial water molecules to approach efficient and reliable prediction of binding free energy. Communicated by Ramaswamy H. Sarma

Hydroxide Ion Mechanism for Long-Range Proton Pumping in the Third Proton Transfer of Bacteriorhodopsin.

In bacteriorhodopsin, representative light-driven proton pump, five proton transfers yield vectorial active proton translocation, resulting in a proton gradient in microbes. Third proton transfer occurs from Asp96 to the Schiff base on the photocycle, which is expected to be a long-range proton transfer via the Grotthuss mechanism through internal water molecules. Here, large-scale quantum molecular dynamics simulations are performed for the third proton transfer, where all the atoms (∼50000 atoms) are treated quantum-mechanically. The simulations demonstrate that two reaction paths exist along the water wire, namely, via hydronium and via hydroxide ions. The free energy analysis confirms that the path via hydroxide ions is considerably favorable and consistent with the observed lifetime of the transient water wire. Therefore, the proposed hydroxide ion mechanism, as in the first proton transfer, is responsible for the third long-range proton transfer.

Towards complete assignment of the infrared spectrum of the protonated water cluster H+(H2O)21

The spectroscopic features of protonated water species in dilute acid solutions have been long sought after for understanding the microscopic behavior of the proton in water with gas-phase water clusters H+(H2O)n extensively studied as bottom-up model systems. We present a new protocol for the calculation of the infrared (IR) spectra of complex systems, which combines the fragment-based Coupled Cluster method and anharmonic vibrational quasi-degenerate perturbation theory, and demonstrate its accuracy towards the complete and accurate assignment of the IR spectrum of the H+(H2O)21 cluster. The site-specific IR spectral signatures reveal two distinct structures for the internal and surface four-coordinated water molecules, which are ice-like and liquid-like, respectively. The effect of inter-molecular interaction between water molecules is addressed, and the vibrational resonance is found between the O-H stretching fundamental and the bending overtone of the nearest neighboring water molecule. The revelation of the spectral signature of the excess proton offers deeper insight into the nature of charge accommodation in the extended hydrogen-bonding network underpinning this aqueous cluster.

Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor.

There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3 ) have been determined by cryogenic electron microscopy (cryo-EM) in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

Anharmonic Vibrational Calculations Based on Group-Localized Coordinates: Applications to Internal Water Molecules in Bacteriorhodopsin.

An efficient anharmonic vibrational method is developed exploiting the locality of molecular vibration. Vibrational coordinates localized to a group of atoms are employed to divide the potential energy surface (PES) of a system into intra- and inter-group contributions. Then, the vibrational Schrödinger equation is solved based on a PES, in which the inter-group coupling is truncated at the harmonic level while accounting for the intra-group anharmonicity. The method is applied to a pentagonal hydrogen bond network (HBN) composed of internal water molecules and charged residues in a membrane protein, bacteriorhodopsin. The PES is calculated by the quantum mechanics/molecular mechanics (QM/MM) calculation at the level of B3LYP-D3/aug-cc-pVDZ. The infrared (IR) spectrum is computed using a set of coordinates localized to each water molecule and amino acid residue by second-order vibrational quasi-degenerate perturbation theory (VQDPT2). Benchmark calculations show that the proposed method yields the N-D/O-D stretching frequencies with an error of 7 cm-1 at the cost reduced by more than five times. In contrast, the harmonic approximation results in a severe error of 150 cm-1. Furthermore, the size of QM regions is carefully assessed to find that the QM regions should include not only the pentagonal HBN itself but also its HB partners. VQDPT2 calculations starting from transient structures obtained by molecular dynamics simulations have shown that the structural sampling has a significant impact on the calculated IR spectrum. The incorporation of anharmonicity, sufficiently large QM regions, and structural samplings are of essential importance to reproduce the experimental IR spectrum. The computational spectrum paves the way for decoding the IR signal of strong HBNs and helps elucidate their functional roles in biomolecules.

Inverse Hydrogen-Bonding Change Between the Protonated Retinal Schiff Base and Water Molecules upon Photoisomerization in Heliorhodopsin 48C12.

Heliorhodopsin (HeR) is a new class of the rhodopsin family discovered in 2018 through functional metagenomic analysis (named 48C12). Similar to typical microbial rhodopsins, HeR possesses seven transmembrane (TM) α-helices and an all-trans-retinal covalently bonded to the lysine residue on TM7 via a protonated Schiff base. Remarkably, the HeR membrane topology is inverted compared with that of typical microbial rhodopsins. The X-ray crystal structure of HeR 48C12 was elucidated after the first report on a HeR variant from Thermoplasmatales archaeon SG8-52-1, which revealed the water-mediated hydrogen-bonding network connected to the Schiff base region in the cytoplasmic side. Herein, low-temperature light-induced FTIR spectroscopic analyses of HeR 48C12 and 15N isotopically labeled proteins were used to elucidate the structural changes during retinal photoisomerization. N-D stretching vibrations of the protonated retinal Schiff base (PRSB) at 2286 and 2302 cm-1 in the dark state, and 2239 and 2252 cm-1 in the K intermediate were observed. The frequency changes indicated that the hydrogen bond of PRSB strengthens upon photoisomerization in HeR. Moreover, O-D stretching vibration frequencies of the internal water molecules indicate that the hydrogen-bonding strength decreases concomitantly. Therefore, the PRSB hydrogen bond responds to photoisomerization in an opposite way to the hydrogen-bonding network involving water molecules. No frequency changes of the indole N-H or N-D stretching vibrations of tryptophan residues were observed upon photoisomerization, suggesting that all tryptophan residues in the HeR 48C12 maintained the hydrogen-bonding strengths in the K intermediate. These results provide insights into the molecular mechanism of the energy storage and propagation upon retinal photoisomerization in HeR.

HomolWat: a web server tool to incorporate 'homologous' water molecules into GPCR structures.

Internal water molecules play an essential role in the structure and function of membrane proteins including G protein-coupled receptors (GPCRs). However, technical limitations severely influence the number and certainty of observed water molecules in 3D structures. This may compromise the accuracy of further structural studies such as docking calculations or molecular dynamics simulations. Here we present HomolWat, a web application for incorporating water molecules into GPCR structures by using template-based modelling of homologous water molecules obtained from high-resolution structures. While there are various tools available to predict the positions of internal waters using energy-based methods, the approach of borrowing lacking water molecules from homologous GPCR structures makes HomolWat unique. The tool can incorporate water molecules into a protein structure in about a minute with around 85% of water recovery. The web server is freely available at http://lmc.uab.es/homolwat.

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds.

Infrared difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods, it stands out by its sensitivity to the protonation state, H-bonding, and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water molecules, or cofactors. In particular, the detection of protonation and H-bonding changes in a time-resolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution, I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the preparation of suitable samples and their characterization, strategies for the perturbation of proteins, and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focuses on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities, and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and complemented by integration and interpretation of the results in the context of the studied protein, an aspect increasingly supported by spectral calculations. Selected examples from the literature, predominately but not exclusively from retinal proteins, are used to illustrate the topics covered in this review.

Effects of material characteristics on the structural characteristics and flavor substances retention of meat analogs

The production of meat analogs using high moisture extrusion cooking is a complex process that depends on the properties of the protein ingredients. The aim of the study was to investigate the effect of different moisture and wheat gluten contents on the retention of volatile flavor substances, microstructure, moisture distribution, and secondary protein structures of meat analogs produced by the high moisture extrusion process. The high moisture extrusion trials were carried out using a soy protein concentrate with 0%, 10%, 20%, 30, and 40% added wheat gluten (by dry mass) or 50%, 60%, 70, and 80% moisture. The comparisons revealed that the retention rates of volatile flavor substances from large to small were generally represented by esters, alkanes, alkenes, phenols, aldehydes, and alcohols, under the same extrusion conditions. The wheat gluten and moisture contents affected the flavor characteristics of the meat analogs by affecting the microstructure, the binding capacity of internal proteins and water molecules, and the secondary structure of the proteins. The higher retention rates of the volatile flavor substances were observed in meat analogs produced by raw material with higher wheat gluten content and lower moisture content. Our findings show that wheat gluten and moisture contents of the raw material regulate the retention rate of volatile flavor substances by affecting the microstructure, water distribution, protein structure, and interactive force of the meat analogs, opening up a wide range of products for different consumer requirements.

An assessment of water placement algorithms in quantum mechanics/molecular mechanics modeling: the case of rhodopsins' first spectral absorption band maxima.

Quantum mechanics/molecular mechanics (QM/MM) models are a widely used tool to obtain detailed insight into the properties and functioning of proteins. The outcome of QM/MM studies heavily depends on the quality of the applied QM/MM model. Prediction and right placement of internal water molecules in protein cavities is one of the critical parts of any QM/MM model construction. Herein, we performed a systematic study of four protein hydration algorithms. We tested these algorithms for their ability to predict X-ray-resolved water molecules for a set of membrane photosensitive rhodopsin proteins, as well as the influence of the applied water placement algorithms on the QM/MM calculated absorption maxima (λmax) of these proteins. We used 49 rhodopsins and their intermediates with available X-ray structures as the test set. We found that a proper choice of hydration algorithms and setups is needed to predict functionally important water molecules in the chromophore-binding cavity of rhodopsins, such as the water cluster in the N-H region of bacteriorhodopsin or two water molecules in the binding pocket of bovine visual rhodopsin. The QM/MM calculated λmax of rhodopsins is also quite sensitive to the applied protein hydration protocols. The best methodology allows obtaining an 18.0 nm average value for the absolute deviation of the calculated λmax from the experimental λmax. Although the major effect of water molecules on λmax originates from the water molecules located in the binding pocket, the water molecules outside the binding pocket also affect the calculated λmax mainly by causing a reorganization of the protein structure. The results reported in this study can be used for the evaluation and further development of hydration methodologies, in general, and rhodopsin QM/MM models, in particular.

Enthalpically-driven ligand recognition and cavity solvation of bovine odorant binding protein

Lipocalins are a widely distributed family of extracellular proteins typically involved in the transport of small hydrophobic molecules. To gain new insights into the molecular basis that governs ligand recognition by this ancient protein family, the binding properties of the domain-swapped dimer bovine odorant binding protein (bOBP) and its monomeric mutant bOBP121G+ were characterized using calorimetric techniques and molecular dynamics simulations. Thermal unfolding profiles revealed that the isolated bOBP subunits behave as a cooperative folding unit. In addition, bOBP and bOBP121G+ exhibited similar ligand binding properties, characterized by a non-classical hydrophobic effect signature. The energetic differences in the binding of bOBP to 1-hexen-3-ol and the physiological ligand 1-octen-3-ol were strikingly larger than those observed for the interaction of other lipocalins with congeneric ligands. MD simulations revealed that the recurrent opening of transient pores in the submicrosecond timescale allows a profuse exchange of water molecules between the protein interior and the surrounding solvent. This picture contrasts with other lipocalins whose ligand-free binding cavities are devoid of solvent molecules. Furthermore, the simulations indicated that internal water molecules solvate the protein cavity suboptimally, forming fewer hydrogen bonds and having lower density and higher potential energy than bulk water molecules. Upon ligand occupation, water molecules were displaced from the binding cavity in an amount that depended on the ligand size. Taken together, calorimetric and MD-simulation results are consistent with a significant contribution of cavity desolvation to the enthalpically-driven interaction of bOBP with its hydrophobic ligands.

Multidomain Convergence of Argonaute during RISC Assembly Correlates with the Formation of Internal Water Clusters

Despite the relevance of Argonaute proteins in RNA silencing, little is known about the structural steps of small RNA loading to form RNA-induced silencing complexes (RISCs). We report the 1.9Å crystal structure of human Argonaute4 with guide RNA. Comparison with the previously determined apo structure of Neurospora crassa QDE2 revealed that the PIWI domain has two subdomains. Binding of guide RNA fastens the subdomains, thereby rearranging the active-site residues and increasing the affinity for TNRC6 proteins. We also identified two water pockets beneath the nucleic acid-binding channel that appeared to stabilize the mature RISC. Indeed, mutating the water-pocket residues of Argonaute2 and Argonaute4 compromised RISC assembly. Simulations predict that internal water molecules are exchangeable with the bulk solvent but always occupy specific positions at the domain interfaces. These results suggest that after guide RNA-driven conformational changes, water-mediated hydrogen-bonding networks tie together the converged domains to complete the functional RISC structure.