The Importance of Structured Water in HDAC8 for Correct Binding Pose Applied for Drug Design of Anticancer Molecules.

AIMS: Validating the docking procedure and maintaining the structural water molecules at HDAC8 catalytic site. BACKGROUND: Molecular docking simulations play a significant role in Computer-Aided Drug Design, contributing to the development of new molecules. To ensure the reliability of these simulations, a validation process called "Re-docking" is employed, focusing on the binding mode of a ligand co-crystallized with the protein of interest. OBJECTIVE: In this study, several molecular docking studies were conducted using five X-ray structures of HDAC8-ligand complexes from the PDB. METHOD: Ligands initially complexed with HDAC8 were removed and re-docked onto the free protein, revealing a poor reproduction of the expected binding mode. In response to this, we observed that most HDAC8-ligand complexes contained one to two water molecules in the catalytic site, which were crucial for maintaining the cocrystallized ligand. RESULT: These water molecules enhance the binding mode of the co-crystallized ligand by stabilizing the proteinligand complex through hydrogen bond interactions between ligand and water molecules. Notably, these interactions are lost if water molecules are removed, as is often done in classical docking methodologies. Considering this, molecular docking simulations were repeated, both with and without one or two conserved water molecules near Zn+2 in the catalytic cavity. Simulations indicated that replicating the native binding pose of co-crystallized ligands on free HDAC8 without these water molecules was challenging, showing greater coordinate displacements (RMSD) compared to those including conserved water molecules from crystals. CONCLUSION: The study highlighted the importance of conserved water molecules within the active site, as their presence significantly influenced the successful reproduction of the ligands' native binding modes. The results suggest an optimal molecular docking procedure for validating methods suitable for filtering new HDAC8 inhibitors for future experimental assays.

Effect of a magnetic field on droplet freezing and frost formation on cold surfaces.

The effect of a magnetic field on condensed droplet freezing and frost formation was investigated using visualized experimental devices in this study. The size, shape, freezing time of droplets, and frost shape on the magnetic field considerably differ from those on the nonmagnetic surface. Moreover, the magnetic field could suppress droplet freezing and frost formation. The magnetic field suppression effect on droplet freezing and frost formation was analyzed according to the polarity characteristics of water molecules.

Concurrent Mechanisms of Hot Electrons and Interfacial Water Molecule Ordering in Plasmon-Enhanced Nitrogen Fixation.

The participation of high-energy hot electrons generated from the non-radiative decay of localized surface plasmons is an important mechanism for promoting catalytic processes. Herein, another vital mechanism associated with the localized surface plasmon resonance (LSPR) effect, significantly contributing to the nitrogen reduction reaction (NRR), is found. That is to say, the LSPR-induced strong localized electric fields can weaken the intermolecular hydrogen bonds and regulate the arrangement of water molecules at the solid-liquid interface. The AuCu pentacle nanoparticles with excellent light absorption ability and the capability to generate strong localized electric fields are chosen to demonstrate this effect. The in situ Raman spectra and theoretical calculations are employed to verify the mechanism at the molecular scale in a nitrogen fixation process. Meanwhile, due to the promoted electron transfer at the interface by the well-ordered interfacial water, as well as the participation of high-energy hot electrons, the optimal catalyst exhibits excellent performance with an NH3 yield of 52.09 µg h-1 cm-2 and Faradaic efficiency (FE) of 45.82% at ─0.20 V versus RHE. The results are significant for understanding the LSPR effect in catalysis and provide a new approach for regulating the reaction process.

Theoretical Analysis of the Influence of Spacers on Salt Ion Transport in Electromembrane Systems Considering the Main Coupled Effects.

This article considers a theoretical analysis of the influence of the main coupled effects and spacers on the transfer of salt ions in electromembrane systems (EMS) using a 2D mathematical model of the transfer process in a desalting channel with spacers based on boundary value problems for the coupled system of Nernst-Planck-Poisson and Navier-Stokes equations. The basic patterns of salt ion transport have been established, taking into account diffusion, electromigration, forced convection, electroconvection, dissociation/recombination reactions of water molecules, as well as spacers located inside the desalting channel. It has been shown that spacers and taking into account the dissociation/recombination reaction of water molecules significantly change both the formation and development of electroconvection. This article confirms the fact of the exaltation of the limiting current studied by Harkatz, where it is shown that the current (flux) of salt ions increases when the dissociation reaction begins by a certain value called the exaltation current, which is proportional to the flow of water dissociation products. A significant combined effect of electroconvection and dissociation/recombination reactions as well as the spacer system in the desalting channel on the transport of salt ions are shown. The complex, nonlinear, and non-stationary interaction of all the main effects of concentration polarization and spacers in the desalting channel are also considered in the work.

Air/Liquid Interfacial Self-Assembled Intrinsically Stretchable IDT-BT Film Combining a Deliberate Transfer Adherence Strategy for Stretchable Electronics.

Indacenodithiophene-benzothiadiazole (IDT-BT) has emerged as one of the most promising candidates for stretchable electronics due to its good stretchability and high mobility. Here, we present an air/liquid interface self-assembly method for the stretchable IDT-BT films and design an air-side transfer adherence strategy for improving the carrier mobility of IDT-BT. By controlling the cosolvent ratio in solution and the solvent evaporation rate, the large-scale intrinsically stretchable IDT-BT film with the diameter as high as ∼3 cm was self-assembled at the air/liquid interface. The resulting stretchable film with lightweight and good uniformity could be easily transferred to curved objects such as flexible 3 M tape, glass ball, and seashell. It is found that the transfer adherence strategy of the semiconductor film significantly affects the carrier transport. The transfer adherence from air-side can effectively decrease the number of the adsorbed water molecules at semiconductor/dielectric interface, which presents the mobility as high as 2.98 cm2 V-1 s-1. Based on the air/liquid interface self-assembled IDT-BT film, the peeling process of the film for preparation of full stretchable transistors could be eliminated. The resulting intrinsically stretchable transistor exhibits mobility higher than that of the transistor with a conventional spin-coated film. Our research provides new pathways for preparing the stretchable films and intrinsically stretchable organic field-effect transistors and shows the promising potential of the air/liquid interface self-assembly strategy for stretchable electronics.

Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center.

The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

Structural water molecules dominated p band intermediate states as a unified model for the origin on the photoluminescence emission of noble metal nanoclusters: from monolayer protected clusters to cage confined nanoclusters.

In the past several decades, noble metal nanoclusters (NMNCs) have been developed as an emerging class of luminescent materials due to their superior photo-stability and biocompatibility, but their luminous quantum yield is relatively low and the physical origin of the bright photoluminescence (PL) of NMNCs remain elusive, which limited their practical application. As the well-defined structure and composition of NMNCs have been determined, in this mini-review, the effect of each component (metal core, ligand shell and interfacial water) on their PL properties and corresponded working mechanism were comprehensively introduced, and a model that structural water molecules dominated p band intermediate state was proposed to give a unified understanding on the PL mechanism of NMNCs and a further perspective to the future developments of NMNCs by revisiting the development of our studies on the PL mechanism of NMNCs in the past decade.

The First-Water-Layer Evolution at the Graphene/Water Interface under Different Electro-Modulated Hydrophilic Conditions Observed by Suspended/Supported Field-Effect-Device Architectures.

Interfacial water molecules affect carrier transportation within graphene and related applications. Without proper tools, however, most of the previous works focus on simulation modeling rather than experimental validation. To overcome this obstacle, a series of graphene field-effect transistors (GFETs) with suspended (substrate-free, SF) and supported (oxide-supported, OS) configurations are developed to investigate the graphene-water interface under different hydrophilic conditions. With deionized water environments, in our experiments, the electrical transportation behaviors of the graphene mainly originate from the evolution of the interfacial water-molecule arrangement. Also, these current-voltage behaviors can be used to elucidate the first-water layer at the graphene-water interface. For SF-GFET, our experimental results show positive hysteresis in electrical transportation. These imply highly ordered interfacial water molecules with a separated-ionic distributed structure. For OS-GFET, on the contrary, the negative hysteresis shows the formation of the hydrogen-bond interaction between the interfacial water layer and the SiO2 substrate under the graphene. This interaction further promotes current conduction through the graphene/water interface. In addition, the net current-voltage relationship also indicates the energy required to change the orientation of the first-layer water molecules during electro-potential change. Therefore, our work gives an insight into graphene-water interfacial evolution with field-effect modulation. Furthermore, this experimental architecture also paves the way for investigating 2D solid-liquid interfacial features.

Computational investigation of functional water molecules in GPCRs bound to G protein or arrestin.

G protein-coupled receptors (GPCRs) are membrane proteins constituting the largest family of drug targets. The activated GPCR binds either the heterotrimeric G proteins or arrestin through its activation cycle. Water molecules have been reported to play a role in GPCR activation. Nevertheless, reported studies are focused on the hydrophobic helical bundle region. How water molecules function in GPCR bound either G protein or arrestin is rarely studied. To address this issue, we carried out computational studies on water molecules in both GPCR/G protein complexes and GPCR/arrestin complexes. Using inhomogeneous fluid theory (IFT), we locate all possible hydration sites in GPCRs binding either to G protein or arrestin. We observe that the number of water molecules on the interaction surface between GPCRs and signal proteins are correlated with the insertion depths of the α5-helix from G-protein or "finger loop" from arrestin in GPCRs. In three out of the four simulation pairs, the interfaces of Rhodopsin, M2R and NTSR1 in the G protein-associated systems show more water-mediated hydrogen-bond networks when compared to these in arrestin-associated systems. This reflects that more functionally relevant water molecules may probably be attracted in G protein-associated structures than that in arrestin-associated structures. Moreover, we find the water-mediated interaction networks throughout the NPxxY region and the orthosteric pocket, which may be a key for GPCR activation. Reported studies show that non-biased agonist, which can trigger both GPCR-G protein and GPCR-arrestin activation signal, can result in pharmacologically toxicities. Our comprehensive studies of the hydration sites in GPCR/G protein complexes and GPCR/arrestin complexes may provide important insights in the design of G-protein biased agonists.

Structural insight to human Retinoid X receptor alpha-Thyroid hormone receptor beta heterodimer by molecular modelling and MD-simulation studies: role of conserved water molecules.

The Retinoid X receptor alpha-Thyroid hormone receptor beta (RXRα-THRß) heterodimer plays an important role in physiological function of humans specially in the growth and development. Extensive MD-simulation studies on the aquated complexes of modelled RXRα-THRß heterodimer with DNA-duplex have indicated the role of some conserved/semiconserved water molecules in the complexation process in presence or absence of Triiodothyronine (T3) and 9-cis retinoic acid (9CR) in the respective Ligand Binding Domain (LBD) domain. Among the seventeen conserved/semi-conserved water molecules, the W1-W4 water centers have been observed to mediate the interaction between the residues of A-chain (DBD of RXR) to consensus sequence (C-chain) of DNA. The W5-W8 water centers involve in recognition of the residues of B-chain (DBD of THR) to C-chain of DNA. The W9-W13 centers have connected the different residues of B-chain (THR) to D-chain of DNA through H-bonds, whereas W14-W17 water molecules were involved in the interaction of A-chain's (RXR) residues to D-chain of DNA. In our previous study with homodimeric THRß from Rattus norvegicus we have identified fifteen conserved water molecules at the DNA-DBD interface. Moreover, the conformational flexibility of Met313 (in the LBD of THR) from open to close form in presence or absence of T3 molecule in the holo and Apo-protein may provide a plausible rational on the possible role of that residue to acts as gate which could restrict the solvent molecules to enter into the hydrophobic T3-binding pocket of LBD during the absence of ligand molecule and thus could help the stabilization of that domain in THRß structure.Communicated by Ramaswamy H. Sarma.

Evaporation of water and urea solution in a magnetic field; the role of nuclear isomers.

HYPOTHESIS: Ortho and para water are the two nuclear isomers where the hydrogen protons align to give a total nuclear spin of 1 or 0. The equilibrium ratio of 3:1 is established slowly in freshly evaporated water vapour while the isomers behave distinct gasses, with their own partial pressures. Magnetic-field-induced ortho âŸ· para transformations are expected to alter the evaporation rate. EXPERIMENT: Evaporation from beakers of deionized water and a 6 M solution of urea is monitored simultaneously for periods from 1 to 60 h with and without a 500 mT magnetic field, while logging the ambient temperature and humidity. Balances with the two beakers are shielded in the same Perspex container. Many runs have been conducted over a two-year period. FINDINGS: The evaporation rate of water is found to increase by 12 ± 7% of in the field but that of water with dissolved urea decreases by 28 ± 6%. Two effects are at play. One is dephasing of the Larmor precession of adjacent protons on a water molecule in a field gradient, which tends to equalize the isomer populations. The other is Lorentz stress on the moving charge dipole, which can increase the proportion of the ortho isomer. From analysis of the time and field dependence of the evaporation, we infer that the ortho fraction is 39 ± 1% in fresh vapour from water and 60 ± 5% in fresh vapour from urea.

Changes in hydration of liposome membranes exposed to nanosecond electric pulses detected by wide-field Coherent anti-Stokes Raman microspectroscopy.

Electropulsation has become a powerful technological platform for electromanipulation of cells and tissues for various medical and biotechnological applications, but the molecular changes that underlay the very first initiation step of this process have not been experimentally observed. Here, we endowed a wide-field Coherent anti-Stokes Raman Scattering platform with an ad-hoc electromagnetic exposure device and we demonstrated, using artificial lipid vesicles (i.e. liposomes), that electropulsation is initiated by the increase of interstitial water content in liposome membranes. A pulse-dependent accumulation of the interstitial water molecules is observed in the membranes and a plausible mechanism supported by a computational electrochemical model is presented and discussed.

Hydration effect of selected atmospheric gases with finite water clusters: A quantum chemical investigation towards atmospheric implications.

Water vapor in atmosphere is ubiquitous, and it varies according to geographical locations. Various toxic and non-toxic gases co-exist with water vapor/moisture in the atmosphere. This computational study addresses the fact that how those gases interact with water vapor. We have done quantum chemical density functional theory calculations to probe the interaction of certain gases with a finite number of water molecules in gas phase with various functionals/basis sets. An ensemble of 14 gas molecules comprising various diatomic, triatomic, and polyatomic gases have been chosen for the investigations. The intermolecular interactions are understood from the interaction energy, electrostatic potential, frontier molecular orbitals, energy gap, and natural bond orbital analyses. Furthermore, quantum molecular descriptors such as electronegativity, chemical potential, chemical hardness and electrophilicity index are calculated to have deep insight on chemical nature of the gas molecules. Additionally, we have done implicit solvent modelling using PCM, and the corresponding solvation energies have been calculated. Interestingly, all the calculations and analyses have projected the similar results that Cl2, SO2, and NH3 have very high interaction with the water clusters. To mimic various altitudes (0 km, 5 km and 10 km) in the atmosphere, thermochemistry calculations have been carried out at different temperature and pressure values. The Gibbs free energies of formation suggest that the hydration of Cl2 is higher followed by O2, SO2 and NH3 at all altitudes. Remarkably, it is found that the formation of hydrated clusters of Cl2 and O2 with 4H2O are thermodynamically favourable. On the other hand, SO2 and NH3 requires 5H2O and 3H2O to form thermodynamically favourable clusters. In summary, it is anticipated that this kind of extensive computational studies facilitate to understand the structural, electronic, chemical and thermochemical properties of hydrated atmospheric gases that leads to the formation of prenucleation clusters followed by atmospheric aerosols.

Molecular modelling and computational studies of peptide diphenylalanine nanotubes, containing waters: structural and interactions analysis.

The work is devoted to computer studies of the structural and physical properties of such self-organizing structures as peptide nanotubes (PNT) based on diphenylalanine (FF) dipeptide with different initial isomers of the left (L-FF) and right (D-FF) chiralities of these dipeptides. The structures under study are considered both with empty anhydrous and with internal cavities filled with water molecules. Molecular models of both chiralities are investigated using quantum-chemical DFT and semi-empirical methods, which are in consistent with the known experimental data. To study the effect of nano-sized clusters of water molecules embedded in the inner hydrophilic cavity on the properties of nanotubes (including the changes in their dipole moments and polarizations), as well as the changes in the structure and properties of water clusters themselves (their own dipole moments and polarizations), the surfaces of internal cavities of nanotubes and outer surfaces of water cluster structures for both types of chirality are analyzed. A specially developed method of visual differential analysis of structural features of (bio)macromolecular structures is applied for these studies. The results obtained of a number of physical properties (interacting energies, dipole moments, polarization values) are given for various cases and analyzed in comparison with the known data. These data are necessary for analyzing the interactions of water molecules with hydrophilic parts of nanotube molecules based on FF, such as COO- and NH3 + , since they determine many properties of the structures under study. The data obtained are useful for further analysis of the possible adhesion and capture of medical molecular components by active layers of FF-based PNT, which can be designed for creating capsules for targeted delivery of pharmaceuticals and drugs on their basis.

Ribose and Non-Ribose A2A Adenosine Receptor Agonists: Do They Share the Same Receptor Recognition Mechanism?

Adenosine receptors have been a promising class of targets for the development of new therapies for several diseases. In recent years, a renewed interest in this field has risen, thanks to the implementation of a novel class of agonists that lack the ribose moiety, once considered essential for the agonistic profile. Recently, an X-ray crystal structure of the A2A adenosine receptor has been solved, providing insights about the receptor activation from this novel class of agonists. Starting from this structural information, we have performed supervised molecular dynamics (SuMD) simulations to investigate the binding pathway of a non-nucleoside adenosine receptor agonist as well as one of three classic agonists. Furthermore, we analyzed the possible role of water molecules in receptor activation.

Insights on the proton translocation pathways in FоF1-ATP synthase using molecular dynamics simulations.

Proton translocation through the Fo fraction of FoF1-ATP synthase is one of the crucial processes in the catalytic cycle of the enzyme. However, the exact trace of protons movement has not been finally established yet because the location and structure of the half-channels are still the subject of investigation. We described the possible network of polar amino acids residues and water molecules that can favor the preferential proton pathway using molecular dynamics simulation of the membrane part of the E. coli ATP synthase embedded in the lipid bilayer and water environment. The inlet half-channel was a complex structure with two entrances in the form of aqueous lacunae and a highly conservative proton transfer chain near Asp61 of c-subunit including amino acids residues and three structural water molecules (W1-W3), while the outlet half-channel was just a water cavity through which a proton can easily move into the cytoplasm. Moreover, the side chains of Asn214 and Gln252 of a-subunit had the stable spatial positions (SP1-SP3). аAsn214 in position SP3 and аGln252 in SP1, SP2 were oriented towards cAsp61 and could presumably protonate it via W1. Herewith aAsn214 in SP1, SP2 was oriented to aHis245. Thus, the proton transfer chain is always unclosed, and switching between positions SP1/SP2 and SP3 of aAsn214 determines the time of proton transport and the movement in this region is the rate-limiting step. In addition, we found another rare position SP3, in which aGln252 is oriented to aAsn116 and aSer144, located outside of the "main H+ route" and being a dead end. The new findings would help to evaluate the whole process of the proton translocation through FoF1-ATP synthase.

Water-mediated structural rearrangement establishes active conformation of caspases for apoptosis and inflammation.

Caspases are cysteine-dependent aspartate-specific proteases that play a crucial role in apoptosis (or programmed cell death) and inflammation. Based on their function, caspases are majorly categorized into apoptotic (initiator/apical and effector/executioner) and inflammatory caspases. Caspases undergo transition from an inactive zymogen to an active caspase to accomplish their function. This transition demands structural rearrangements which are most prominent at the active site loops and are imperative for the catalytic activity of caspases. In effector caspase-3, the structural rearrangement in the active site loop is shown to be facilitated by a set of invariant water (IW) molecules. However, the atomic details involving their role in stabilizing the active conformation have not been reported yet. Moreover, it is not known whether water molecules are essential for the active conformation in all caspases. Thus, in this study, we located IW molecules in initiator, effector, and inflammatory caspases to understand their precise role in rendering the structural arrangement of active caspases. Furthermore, IW molecules involved in anchoring the fragments of the protomer and rendering regulated flaccidity to caspases were identified. Location and identification of IW molecules interacting with amino acid residues involved in establishing the active conformation in the caspases might facilitate the design of potent inhibitors during up-regulated caspase activity in neurodegenerative and immune disorders. Communicated by Ramaswamy H. Sarma.

Pivotal role of water molecules in the photodegradation of pymetrozine: New insights for developing green pesticides.

Photodegradation of the insecticide pymetrozine (PYM) was studied on surface of wax films, and in aqueous and nonaqueous phase. The half-life of PYM on the wax surface was approximately 250 times longer than in water. Scavenging experiments, laser flash photolysis, and spectra analysis indicated the first singlet excited state of PYM (S1 *PYM) to be the most important photoinduced species initiating the photodegradation. Quantum chemistry calculations identified significant molecular torsion and changes in the structure C-CN-N of S1 *PYM, and the absolute charges of the CN atoms increased and the bond strength weakened. Free energy surface analysis, and O18 labeling experiments further confirmed that the mechanism was two-step photoinduced hydrolysis. The first step is the hydrolysis of S1 *PYM at CN upon reaction with 2-3 water molecules (one H2O molecule as the catalyst). The second step is an intramolecular hydrogen transfer coupled with the cleavage of C-N bond and formation of two cyclic products. During the interactions, water molecules experience catalytic activation by transferring protons, while there is a negligible solvent effect. Clarifying the detailed photodegradation mechanisms of PYM is beneficial for the development of green pesticides that are photostable and effective on leaf surfaces, and photolabile and detoxified in the aquatic environment.

New biochemical insights into the dynamics of water molecules at the GMP or IMP binding site of human GMPR enzyme: A molecular dynamics study.

Human GMP reductase (hGMPR) enzyme is involved in a cellular metabolic pathway, converting GMP into IMP, and also it is an important target for anti-leukemic agents. Present computational investigations explain dynamical behavior of water molecules during the conformational transition process from GMP to IMP using molecular dynamics simulations. Residues at substrate-binding site of cancerous protein (PDB Id. 2C6Q) are mostly more dynamic in nature than the normal protein (PDB Id. 2BLE). Nineteen conserved water molecules are identified at the GMP/IMP binding site and are classified as (i) conserved stable dynamic and (ii) infrequent dynamic. Water molecules W11, W14, and W16 are classified as conserved stable dynamic due to their immobile character, whereas remaining water molecules (W1, W2, W3, W4, W5, W7, W8, W9, W10, W12, W13, W15, W17, W18, and W19) are infrequent with dynamic nature. Entrance or displacement of these infrequent water molecules at GMP/IMP sites may occur due to forward and backward movement of reference residues involving ligands. Four water molecules of hGMPR-I and nine water molecules of hGMPR-II are observed in repetitive transitions from GMP to IMP pathway, which indicates discrimination between two isoforms of hGMPRs. Water molecules in cancerous protein are more dynamic and unstable compared to normal protein. These water molecules execute rare dynamical events at GMP binding site and could assist in detailed understanding of conformational transitions that influence the hGMPR's biological functionality. The present study should be of interest to the experimental community engaged in leukemia research and drug discovery for CML cancer.

Dentin interaction with universal adhesive containing isopropanol solvent studied by solid-state NMR spectroscopy.

OBJECTIVE: This study investigated the chemical and structural changes in the mineral phase and collagen of dentin during application of a mild universal adhesive. Particular attention was paid to the role of isopropanol and changes in water molecules. METHODS: In vitro application of the mild universal adhesive on dentin with two established etching modes (self-etch and etch-and-rinse) was studied using solid state nuclear magnetic resonance spectroscopy. RESULTS: It was evidenced that the etch-and-rinse mode leads to a decrease of the inorganic apatite and a reorganization of the residual mineral phase with a low amount of adhesive phosphate monoesters calcium salt formed, compared to the self-etch mode. In contrast, the adhesive interacts very similarly to the level of dentin collagen in both protocols, with a strong decrease in the amount of the free water molecules induced by the presence of isopropanol as the adhesive solvent, but without significant changes in the initial collagen structure. For both modes, the adhesive acrylates monomers remain mobile and can infiltrate the collagen. SIGNIFICANCE: Understanding the molecular interactions between dentin and adhesive solutions is a major challenge for designing products that lead to the formation of ideal dentin resin hybrid layer. Notably, one point considered essential is the presence of unbound water which, over time, is associated with a hydrolytic degradation of the organic matrix. Isopropanol, as an adhesive solvent, leads to a decrease in the amount of the less stable water molecules while the water molecules strongly attached to the collagen are retained, thus preserving the collagen structure.