Bound Water Molecules Research Articles

Elucidation of a Thermodynamical Feature Attributed to Substrate Binding to the Prokaryotic H+/Oligopeptide Cotransporter YdgR with Calorimetric Analysis: The Substrate Binding Driven by the Change in Entropy Implies the Release of Bound Water Molecules from the Binding Pocket.

Here, we have elucidated the substrate recognition mechanism by a prokaryotic H+/oligopeptide cotransporter, YdgR, using isothermal titration calorimetry. Under acidic conditions (pH 6.0), the binding of a dipeptide, Val-Ala, to YdgR elicited endothermic enthalpy, which compensated for the increase in entropy due to dipeptide binding. A series of dipeptides were used in the binding titration. The dipeptides represent Val-X and X-Val, where X is Ala, Ser, Val, Tyr, or Phe. Most dipeptides revealed endothermic enthalpy, which was completely compensated by the increase in entropy due to dipeptide binding. The change in enthalpy due to binding correlated well with the change in entropy, whereas the Gibbs free energy involved in the binding of the dipeptide to YdgR remained unchanged irrespective of dipeptide sequences, implying that the binding reaction was driven by entropy, that is, the release of bound water molecules in the binding pocket. It is also important to clarify that, based on the prediction of water molecules in the ligand-binding pocket of YdgR, the release of three bound water molecules in the putative substrate binding pocket occurred through binding to YdgR. In the comparison of Val-X and X-Val dipeptides, the N-terminal region of the binding pocket might contain more bound water molecules than the C-terminal region. In light of these findings, we suggest that bound water molecules might play an important role in substrate recognition and binding by YdgR.

Investigation of N,N′‐Disulfopiperazinium Chlorometallates of Fe(III), Ni(II) and Co(II) as Hybrid Catalysts for the Synthesis of 1,2‐Dihydroquinazoline Derivatives

AbstractThree organic salts of N,N′‐disulfopiperazinium cation with complex metal chloride anions of Fe(III), Ni(II) and Co(II) cations were developed as Brønsted‐Lewis acidic hybrid materials in solid state. Their structural compositions were established by FT‐IR, UV‐Visible, SEM, EDX, Powder XRD and Raman techniques. Thermogravimetric analyses showed removal of physisorbed water around 100 °C for Fe(III) salt in contrast to 150–170 °C for Ni(II) & Co(II) organic salts for strongly bound water molecules in highly crystalline state. Their Brønsted acidities were determined from comparative UV‐visible spectra of 4‐nitroaniline in water in presence of the halometallates while Lewis acidity strength was observed via FT‐IR spectra of these salts in presence of pyridine as probe molecule. The catalytic activity of these acidic organic salts was investigated for three‐component synthesis of 1,2‐dihydroquinazolines as recyclable heterogeneous catalysts.

Characterization of Agarose Gels in Solvent and Non-Solvent Media.

Agarose is known to form a homogeneous thermoreversible gel in an aqueous medium over a critical polymer concentration. The solid-liquid phase transitions are thermoreversible but depend on the molecular structure of the agarose sample tested. The literature has mentioned that agarose gels could remain stable in non-solvents such as acetone or ethanol. However, there has been no characterization of their behavior nor a comparison with the gels formed in a good solvent such as water. In the first step of this article, the structure was characterized using 1H and 13C NMR in both D2O and DMSO-d6 solvents. DMSO is a solvent that dissolves agarose regardless of the temperature. First, we have determined a low yield of methyl substitution on the D-galactose unit. Then, the evolution of the 1H NMR spectrum was monitored as a function of temperature during both increasing and decreasing temperature processes, ranging from 25 to 80 °C. A large thermal hysteresis was obtained and discussed, which aided in the interpretation of rheological behavior. The hysteresis of NMR signals is related to the mobility of the agarose chains, which follows the sol/gel transition depending on the chains' association with H-bonds between water and the -OH groups of agarose for tightly bound water and agarose/agarose in chain packing. In the second step of the study, the water in the agarose gel was exchanged with ethanol, which is a non-solvent for agarose. The resulting gel was stable, and its properties were characterized using rheology and compared to its behavior in aqueous media. The bound water molecules that act as plasticizers were likely removed during the exchange process, resulting in a stronger and more brittle gel in ethanol, with higher thermal stability compared to the aqueous gel. It is the first time that such gel is characterized without phase transition when passing from a good solvent to a non-solvent. This extends the domains of application of agarose.

Unraveling the Reaction Mechanism of Russell's Viper Venom Factor X Activator: A Paradigm for the Reactivity of Zinc Metalloproteinases?

Snake venom metalloproteinases (SVMPs) are important drug targets against snakebite envenoming, the neglected tropical disease with the highest mortality worldwide. Here, we focus on Russell's viper (Daboia russelii), one of the "big four" snakes of the Indian subcontinent that, together, are responsible for ca. 50,000 fatalities annually. The "Russell's viper venom factor X activator" (RVV-X), a highly toxic metalloproteinase, activates the blood coagulation factor X (FX), leading to the prey's abnormal blood clotting and death. Given its tremendous public health impact, the WHO recognized an urgent need to develop efficient, heat-stable, and affordable-for-all small-molecule inhibitors, for which a deep understanding of the mechanisms of action of snake's principal toxins is fundamental. In this study, we determine the catalytic mechanism of RVV-X by using a density functional theory/molecular mechanics (DFT:MM) methodology to calculate its free energy profile. The results showed that the catalytic process takes place via two steps. The first step involves a nucleophilic attack by an in situ generated hydroxide ion on the substrate carbonyl, yielding an activation barrier of 17.7 kcal·mol-1, while the second step corresponds to protonation of the peptide nitrogen and peptide bond cleavage with an energy barrier of 23.1 kcal·mol-1. Our study shows a unique role played by Zn2+ in catalysis by lowering the pKa of the Zn2+-bound water molecule, enough to permit the swift formation of the hydroxide nucleophile through barrierless deprotonation by the formally much less basic Glu140. Without the Zn2+ cofactor, this step would be rate-limiting.

Ultra-long cycle life flexible quasi-solid-state alkaline zinc batteries enabled by PEDOT:PSS encapsulated Ni3S2 nanorods

Alkaline nickel-zinc (Ni-Zn) batteries are considered as one of the most promising rechargeable battery systems. However, the poor cycling stability of the Ni cathode limit their service life. Herein, a conductive polymer (PEDOT:PSS) encapsulated Ni3S2 hollow nanorod structure via facile immersion is proposed. PEDOT:PSS coating layer can not only provide adhesion for Ni3S2 nanorods to prevent Ni3S2 from falling off the substrate during cycling, but also facilitate electron transfer via the complete conductive pathway. The hollow nanorods structure of Ni3S2 is beneficial to mitigate the volume change and accelerate the rapid transport of ions. Besides, a gel electrolyte with double network porous structure in our battery system is well designed. The ionic association of sodium alginate on Zn2+ can effectively reduce the activity of bound water molecules in the solvation sheath of Zn2+, inhibit the side reaction and dendrite generation through the ion-confinement effect, to achieve uniform deposition of zinc ions. As a consequence, the as-designed quasi-solid-state flexible Ni-Zn battery demonstrates a high specific capacity of 1.06 mAh cm−2 (276.04 mAh g−1) at 8 mA cm−2 and an ultra-long cycle stability over 10,000 cycles with a capacity retention of 88.96% at 30 mA cm−2.

Anionic polysaccharides for stabilization and sustained release of antimicrobial peptides

Chemical and enzymatic in vivo degradation of antimicrobial peptides represents a major challenge for their therapeutic use to treat bacterial infections. In this work, anionic polysaccharides were investigated for their ability to increase the chemical stability and achieve sustained release of such peptides. The investigated formulations comprised a combination of antimicrobial peptides (vancomycin (VAN) and daptomycin (DAP)) and anionic polysaccharides (xanthan gum (XA), hyaluronic acid (HA), propylene glycol alginate (PGA) and alginic acid (ALG)). VAN dissolved in buffer of pH 7.4 and incubated at 37 °C showed first order degradation kinetics with a reaction rate constant kobs of 5.5 × 10-2 day−1 corresponding with a half-life of 13.9 days. However, once VAN was present in a XA, HA or PGA-based hydrogel, kobs decreased to (2.1–2.3) × 10-2 day−1 while kobs was not affected in an alginate hydrogel and a dextran solution (5.4 × 10-2 and 4.4 × 10-2 day−1). Under the same conditions, XA and PGA also effectively decreased kobs for DAP (5.6 × 10-2 day−1), whereas ALG had no effect and HA even increased the degradation rate. These results demonstrate that the investigated polysaccharides (except ALG for both peptides and HA for DAP) slowed down the degradation of VAN and DAP. DSC analysis was used to investigate on polysaccharide ability to bind water molecules. Rheological analysis highlighted that the polysaccharides containing VAN displayed an increase in G’ of their formulations, pointing that the peptides interaction act as crosslinker of the polymer chains. The obtained results suggest that the mechanism of stabilization of VAN and DAP against hydrolytic degradation is conferred by electrostatic interactions between the ionizable amine groups of the drugs and the anionic carboxylate groups of the polysaccharides. This, in turn, results in a close proximity of the drugs to the polysaccharide chain, where the water molecules have a lower mobility and, therefore, a lower thermodynamic activity.

ANISOTROPY IN THE ADSORPTION OF H2O ON KAOLINITE SURFACES

Adsorption capacities of kaolinite's edge and basal surfaces may differ. Тhese features determine the nature of its adsorption behavior while remaining poorly studied. At the same time, the anisotropy of the edge surface stipulates the wide application of kaolinite in geochemical, environmental, and technological fields. In this work, ab initio calculations have been employed to determine the effect of kaolinite surface anisotropy on its adsorption capacity. The mechanism has been studied for binding water molecules to the most stable kaolinite surfaces, namely, (001), (001¯), (010), and (110). It has been found that the most energetically advantageous adsorption configurations of water molecules arise on edge surface (010), with adsorption energy being ~four times higher than that on basal surface (001¯). The observed energy difference is due to the fact that hydroxyl groups are involved in the adsorption mechanism in addition to surface oxygen atoms. The absence of these groups on the basal surface significantly reduces the probability of hydrogen bonding. The results obtained are in agreement with the data from published studies.

Enhancement of surimi gel properties through the synergetic effect of fucoidan and oligochitosan

For the first time, two common marine-derived dietary fibres (MDFs), fucoidan (FU) and oligochitosan (OCS), were introduced as textural and nutritional enhancers in hairtail surimi gels. The MDFs could assist with inhabiting the endogenous proteolytic enzyme activity, unfolding the myosin to expose more reactive domains, inducing favorable protein conformational transition, and thus, promoting gelation. The highly hydrophilic MDFs rich in –OH groups can bind water molecules via strong hydrogen bonds, facilitating water redistribution within the gel network. Driven by the enhanced chemical forces, a stable protein-FU-OCS gel is obtained, which improves the hardness by almost 100% and the water holding capacity from 86.25% to 92.25%. Collectively, this study demonstrates that MDFs are a group of effective additives to improve gel characteristics and nutritional profiles of surimi-based seafood products. The proposed MDF-protein interaction model would guide the application of MDFs as novel additives in the food industry.

Polymorphism and Ligand Binding Modulate Fast Dynamics of Human Telomeric G-Quadruplexes

Telomeric G-quadruplexes (G4s) are promising targets in the design and development of anticancer drugs. Their actual topology depends on several factors, resulting in structural polymorphism. In this study, we investigate how the fast dynamics of the telomeric sequence AG3(TTAG3)3 (Tel22) depends on the conformation. By using Fourier transform Infrared spectroscopy, we show that, in the hydrated powder state, Tel22 adopts parallel and mixed antiparallel/parallel topologies in the presence of K+ and Na+ ions, respectively. These conformational differences are reflected in the reduced mobility of Tel22 in Na+ environment in the sub-nanosecond timescale, as probed by elastic incoherent neutron scattering. These findings are consistent with the G4 antiparallel conformation being more stable than the parallel one, possibly due to the presence of ordered hydration water networks. In addition, we study the effect of Tel22 complexation with BRACO19 ligand. Despite the quite similar conformation in the complexed and uncomplexed state, the fast dynamics of Tel22-BRACO19 is enhanced compared to that of Tel22 alone, independently of the ions. We ascribe this effect to the preferential binding of water molecules to Tel22 against the ligand. The present results suggest that the effect of polymorphism and complexation on the G4 fast dynamics is mediated by hydration water.

In-situ investigation of solid phase evolution during lyophilization of mannitol-based antibody formulations using an XRPD climate chamber

Crystalline mannitol is commonly used as bulking agent in antibody formulations to provide structure to the lyophilized cake and prevent collapse. Depending on the lyophilization process conditions mannitol can either crystallize as α-, β-, δ-mannitol, mannitol-hemihydrate, or transition to its amorphous state. While crystalline mannitol helps to create a firmer cake structure this is not true for amorphous mannitol. The hemihydrate is also an undesired physical form as it may reduce the drug product stability by releasing bound water molecules into the cake. Our aim was to simulate lyophilization processes in an X-ray powder diffraction (XRPD) climate chamber. In the climate chamber, the process can be carried out fast with low sample quantities to determine optimal process conditions. Insights on the emergence of desired anhydrous mannitol forms helps to adjust the process parameters in larger scale freeze-dryers. In our study we have identified the critical process steps for our formulations and then varied relevant process parameters, which were the annealing temperature, annealing time and temperature ramp rate of the freeze-drying process. Furthermore, the effect of the presence of antibodies on excipient crystallization was investigated by performing the studies on placebo solutions versus two respective antibody formulations. A comparison of the products obtained in a freeze-dryer and the simulated process in the climate chamber showed good accordance demonstrating the method as suitable tool to identify ideal process conditions on a laboratory scale.

Water adsorption properties of microalgae powders: Thermodynamic analysis and structural characteristics

Moisture adsorption isotherms and thermodynamic properties of dried Chlorella pyrenoidesa and Spirulina platensis powders were investigated by using a static gravimetric at temperatures of 20, 30, and 40 °C and within aw range of 0.112–0.976. Structural characteristics involving morphology, functional group, surface element composition, pore size distribution, and surface area were analyzed to explore the water adsorption mechanism. FTIR and XPS analysis indicated that spirulina powder had a higher proportion of hydrophilic sites to bind water molecules in comparison with chlorella powder. BET surface area for chlorella and spirulina powders were 25.10 and 25.19 m2/g, respectively. Water adsorption data were adjusted using a variety of sorption models, and the GAB equation was the most appropriate for representing the experimental data in the studied aw and temperature range. The monolayer moisture content (Xm) values at 20, 30 and 40 °C were 0.0526, 0.0483, and 0.0456 g/g dry basis for chlorella powders, and 0.0581, 0.0553, and 0.0420 g/g dry basis for spirulina powders, respectively. The net isosteric heat (Qst) and differential entropy (δSdif) for both microalgae powders decreased with an increase in water content. Using the compensation theory, we determined that the processes were governed by the enthalpy and that they were non-spontaneous (δG>0) for the samples. Both microalgae powers were classified as micro and mesoporous materials, and the pore radius (Rp) varied from 1.481 to 23.694 nm for chlorella powders and from 1.193 to 39.392 nm for spirulina powders, respectively.

Synthesis and Self-Assembly of β-Octa[(4-Diethoxyphosphoryl)phenyl]porphyrins.

The β-substituted porphyrinoids commonly used to form functional assembled systems in nature yet are still scarcely used in material chemistry probably due to the laborious synthesis of these compounds. In this work, β-octa[(4-diethoxyphosphoryl)phenyl]porphyrin (2HOPPP) and its metal (Zn(II), Cd(II), Cu(II), and Ni(II)) complexes were prepared in good yields. These highly soluble chromophores were characterized in solution using spectroscopic (NMR, UV-vis, fluorescence), electrochemical, and spectroelectrochemical methods. Attachment of the electron-deficient residue (ArP(O)(OEt)2) to the porphyrin macrocycle leads to easier reductions and harder oxidations of the macrocycle for all complexes studied as compared to corresponding meso-tetra[4-(diethoxyphosphoryl)phenyl]porphyrin derivatives reported previously. We demonstrated that the strong electron-deficient character of the MOPPP porphyrins results principally from the increase in the number of electron-withdrawing groups at the periphery of the tetrapyrrolic macrocycle. Electron-deficient porphyrins are highly required in supramolecular and material chemistry in part due to their ability to form supramolecular assemblies via the coordination of axial ligands to the central metal atom. According to single-crystal X-ray data, ZnOPPP forms in the crystalline phase dimers in which each of the two tetrapyrrolic macrocycles is connected through an unusual combination of hydrogen bonding of two phosphoryl groups and the water molecules axially coordinated to the zinc atom of the partner molecule. The involvement of water molecules in porphyrin binding allows for an increase of distance between two porphyrin mean N4 planes, up to 4.478 Å. The offset of phosphoryl groups attached to the macrocycle through a 1,4-phenylene spacer withdraws the whole porphyrin macrocycle of one molecule from spatial overlap with the macrocycle of a partner molecule and increases the Zn-Zn distance up to 10.372 Å. This still unknown type of porphyrin dimers allows one to get deeper insights into the organization of naturally occurring tetrapyrrolic macrocycles. ZnOPPP also forms a labile dimeric complex in 5.3 × 10-7-5.8 × 10-5 M chloroform solutions. In contrast, other complexes prepared in this work exist as monomeric species under these experimental conditions. The self-association constant of ZnOPPP has been determined by electronic absorption spectroscopy.

Charge regulation indicates water expulsion from silica surface by cesium cations

HypothesisSince the discovery of the Hofmeister effect in 1888, the varied propensity of ions to proteins, DNA and other surfaces has motivated research aimed at deciphering the underlying ion specific adsorption mechanism. Experimental and numerical studies have shown that in agreement with Collins' heuristic law of matching water affinity, weakly hydrated (chaotropic) ions adsorb preferentially to hydrophobic surfaces. Here, we show that this preference is driven by expulsion of bound water molecules from the surface by the adsorbing ions. ExperimentsUsing AFM spectroscopy of the force acting between two silica surfaces, we characterize surface charge regulation by adsorbed Na+ and Cs+ ions at different salt concentrations, pH values and temperatures. These data are analyzed in the framework of a recent theory of charge regulation, relating it to change in surface entropy. FindingsUpon binding to the silica, cesium cations expel water molecules from the surface to create additional adsorption sites for more ions. Cs+ adsorption is thus driven by the release of hydrating water molecules and the resulting increased surface entropy. The model indicates that on average, the binding of three cesium cations releases enough water molecules to make room for two additional bound cations. Na+ does not exhibit such behavior.

Zavitsas’ model of aqueous NaF solution activities utilizing hydration numbers reported from Dielectric Relaxation spectroscopy

Dielectric Relaxation Spectroscopy (DRS) measures the number of water molecules irrotationally bound to electrolytes, which is a similar concept to the hydration number in Zavitsas’ model of electrolyte solution thermodynamics. Zavitsas’ model assumes that some water molecules are bound to electrolytes so strongly that they act as part of the electrolyte rather than the solvent. Most monovalent electrolytes have linear water activity versus concentration plots at low concentrations and many sparingly soluble electrolytes like NaF are only soluble in the linear region. Multiparameter models fit to this data are statistically overparametrized. The present study relieves overparameterization by substituted the number of irrotationally bound water molecules determined by DRS for the hydration number in Zavitsas’ model and then optimized the NaF extent of dissociation (ie) to best fit water activity data. This ie value was consistent with and could be predicted by The Law of Matching Water Affinities. Using the resulting model parameters, the solubility of NaF was predicted in aqueous NaNO3 solutions without having to optimize any parameters to the solubility data. These results demonstrates that DRS can be used to parametrize Zavitsas’ model.

Assembly Rules and Dehydration Mechanism of an Unconventional Hydrate: On the Complexity of the Hydrates of Creatine Phosphate Sodium

Unconventional hydrates with differently bound water molecules and related mazy intermolecular interaction networks need systematic investigation. The assembly rules, interaction analysis, and dehydration behavior can be dramatically more complicated when compared with those of common hydrates. In this work, creatine phosphate sodium (CPS) was selected as a model compound representing unconventional hydrates. The packing mode and the role of water molecules related to the dehydration mechanism were explored by the combination of experimental (diffraction, thermal, microscopy) and quantum chemistry computational (interplay visualization, binding energy calculation) methods. It was observed that in the structure of CPS heptahydrate and heminonahydrate, channel, isolated-site, and ion-associated water molecules exist simultaneously, constructing the framework via the coordination bond and hydrogen bond. The binding energy of some ion-coordinated water molecules is lower than that of hydrogen-bonded ones while that of some isolated-site water molecules is lower when compared with that of channel ones, which is counterintuitive. Moreover, the diverse types and locations of water molecules, complicated H2O···H2O interactions, and the trade-off between CPS···H2O and H2O···H2O lead to multistage, variable, and binding-energy-independent dehydration behavior. This work sheds light on the novel structure and variable dehydration mechanism of complicated hydrate systems and inspires the particularity and further investigation of unconventional hydrates.

Studies on ammonium dinitramide and 3,4-diaminofurazan cocrystal for tuning the hygroscopicity

Ammonium dinitramide (ADN) is a promising oxidizer with high energy characteristic, which is a relatively new environmentally friendly oxidizer without halogens and carbon elements. However, ADN has high hygroscopicity when exposed to high humidity air, restricting its applications on the solid propellants. In this paper, a novel energetic cocrystal composed of ammonium dinitramide and 3,4-diaminofurazan (DAF) was proposed and successfully synthesized by antisolvent crystallization method, and the properties of the cocrystal were systematically investigated by analytical characterization and theoretical simulation calculations. The formation of the cocrystal was confirmed by powder X-ray diffraction, differential scanning calorimetry, scanning electron microscopy, infrared spectroscopy and Raman spectroscopy, indicating that the synthesized product was a cocrystal. Through theoretical studies, the ADN/DAF cocrystal structure was predicted, and the powder X-ray diffraction, morphology, water sorption capacity of ADN/DAF cocrystal were calculated, which was consistent with experimental phenomena. The results showed that newly prepared cocrystal of ADN/DAF had lower hygroscopicity compared to pure ADN, and the water sorption capacity was reduced from 15.35% to 7.90%. This may be due to the formation of NH⋯O medium-strength hydrogen bonds between the ammonium ion of ADN and the O atom of DAF in the cocrystal, which prevents the binding of water molecules in the air and ammonium ions and reduces the probability of ADN binding to water molecules, leading to the reduction of cocrystal hygroscopicity. The newly prepared energetic cocrystal can provide theoretical and technical guidance for the study of the anti-hygroscopicity of ADN and advance the practical application of ADN.

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.

Specific zinc binding to heliorhodopsin.

Heliorhodopsins (HeRs), a recently discovered family of rhodopsins, have an inverted membrane topology compared to animal and microbial rhodopsins. The slow photocycle of HeRs suggests a light-sensor function, although the actual function remains unknown. Although HeRs exhibit no specific binding of monovalent cations or anions, recent ATR-FTIR spectroscopy studies have demonstrated the binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR) and 48C12. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding, suggesting a possible modification of the HeR function by Zn2+. The present study shows that Zn2+-binding lowers the thermal stability of TaHeR, and slows back proton transfer to the retinal Schiff base (M decay) during its photocycle. Zn2+-binding was similarly observed for a TaHeR opsin that lacks the retinal chromophore. We then studied the Zn2+-binding site by means of the ATR-FTIR spectroscopy of site-directed mutants. Among five and four mutants of His and Asp/Glu, respectively, only E150Q exhibited a completely different spectral feature of the α-helix (amide-I) in ATR-FTIR spectroscopy, suggesting that E150 is responsible for Zn2+-binding. Molecular dynamics (MD) simulations built a coordination structure of Zn2+-bound TaHeR, where E150 and protein bound water molecules participate in direct coordination. It was concluded that the specific binding site of Zn2+ is located at the cytoplasmic side of TaHeR, and that Zn2+-binding affects the structure and structural dynamics, possibly modifying the unknown function of TaHeR.

The Ewald sphere/focus gradient does not limit the resolution of cryoEM reconstructions

In our quest to solve biomolecular structures to higher resolutions in cryoEM, care must be taken to deal with all aspects of image formation in the electron microscope. One of these is the Ewald sphere/focus gradient that derives from the scattering geometry in the microscope and its implications for recovering high resolution and handedness information. While several methods to deal with it has been proposed and implemented, there are still questions as to the correct approach. At the high acceleration voltages used for cryoEM, the traditional projection approximation that ignores the Ewald sphere breaks down around 2–3 Å and with large particles. This is likely not crucial for most biologically interesting molecules, but is required to understand detail about catalytic events, molecular orbitals, orientation of bound water molecules, etc. Through simulation I show that integration along the Ewald spheres in frequency space during reconstruction, the “simple insertion method” is adequate to reach resolutions to the Nyquist frequency. Both theory and simulations indicate that the handedness information encoded in such phases is irretrievably lost in the formation of real space images. The conclusion is that correct reconstruction along the Ewald spheres avoids the limitations of the projection approximation.

Molecular Structure and Dynamics in Wet Gecko β-Keratin.

Molecular dynamics simulations are performed to investigate the molecular picture of water sorption in gecko keratin and the influence of relative humidity (RH) on the local structure and dynamics in water-swollen keratin. At low RHs, water sorption occurs through hydrogen bonding of water with the hydrophilic groups of keratin. At high RHs (>80%), additional water molecules connect to the first "layer" of amide-connected water molecules (multimolecular sorption) through hydrogen bonds, giving rise to a sigmoidal shape of the sorption isotherm. This causes the formation of large chain-like clusters surrounding the hydrophilic groups of keratin, which upon a further increase of the RH form a percolating water network. An examination of the dynamics of water molecules sorbed in keratin demonstrates that there are two states, bound and free, for water. The dynamics of water in these states depends on the RH. At low RHs, large-scale translational motions of tightly bound water molecules to keratin are needed to remake the entire hydration shell of the keratin. At high RHs (>80%), the water molecules more quickly exchange between the two states. The center-of-mass mean-square displacement of water molecules indicates a hopping motion of water molecules in the keratin solvation shell. The hopping mechanism is more pronounced at RHs < 80%. At higher RHs, water translation through water clusters (water network) dominates. We have observed two regimes for the dependence of dynamical properties on the RH: a regime of gradual increase of the dynamics over 10% < RH < 80% and a regime of drastic dynamic acceleration at RH > 80%. The latter regime begins exactly where the water uptake and the volume swelling also increase much more and where a drastic change in the elastic properties of gecko keratin has been observed. A nearly linear relation between the relaxation times for all dynamical processes and the water content of gecko keratin is observed.