Insights into structural mechanisms of gating induced regulation of aquaporins

Aquaporin family comprises of transmembrane channels that are specialized in conducting water and certain small, uncharged molecules across cell membranes. Essential roles of aquaporins in various physiological and pathophysiological conditions have attracted great scientific interest. Pioneering structural studies on aquaporins have almost solved the basic question of mechanism of selective water transport through these channels. Another important structural aspect of aquaporins which seeks attention is that how the flow of water through the channel is regulated by the mechanism of gating. Aquaporins are also regulated at the protein level, i.e. by trafficking which includes changes in their expression levels in the membrane. Availability of high resolution structures along with numerous molecular dynamics simulation studies have helped to gain an understanding of the structural mechanisms by which water flux through aquaporins is controlled. This review will summarize the highlights regarding structural features of aquaporins, mechanisms governing water permeation, proton exclusion and substrate specificity, and describe the structural insights into the mechanisms of aquaporin gating whereby water conduction is regulated by post translational modifications, such as phosphorylation.     

Effects of ethanol on the secondary structure specific hydration properties of Chymotrypsin Inhibitor 2 in its folded and unfolded forms

Molecular dynamics simulations were performed for the protein Chymotrypsin Inhibitor 2 in water–ethanol binary mixtures at several ethanol concentrations at two different temperatures to explore the differential role of ethanol on the hydration properties of the secondary structural segments of the protein. We find that, compared to the water structure around the secondary structure segments of the unfolded protein in pure water, water molecules preferred to be structured more in presence of ethanol. The structuration of ethanol is noticed to be higher around helix and sheet, as compared to that around the loop. The differential slow structural relaxations of the hydrogen bonds involving the secondary structural segments of protein in water–ethanol mixed solutions, as compared to that in pure water infer that ethanol alters the water properties in a remarkable manner. The slow relaxations of the hydrogen bonds formed between ethanol and the secondary structural segments of the protein, in general, suggests that ethanol interacts directly with the protein. However, the relatively faster relaxation of the ethanol-segment hydrogen bonds involving unfolded protein as compared to that involving the folded protein suggests that the unfolded protein surface can remain dynamically free to interact with water more.     

Theoretical study of the partial molar volume change associated with the pressure-induced structural transition of ubiquitin

The partial molar volume (PMV) change associated with the pressure-induced structural transition of ubiquitin is analyzed by the three-dimensional reference interaction site model (3D-RISM) theory of molecular solvation. The theory predicts that the PMV decreases upon the structural transition, which is consistent with the experimental observation. The volume decomposition analysis demonstrates that the PMV reduction is primarily caused by the decrease in the volume of structural voids in the protein, which is partially canceled by the volume expansion due to the hydration effects. It is found from further analysis that the PMV reduction is ascribed substantially to the penetration of water molecules into a specific part of the protein. Based on the thermodynamic relation, this result implies that the water penetration causes the pressure-induced structural transition. It supports the water penetration model of pressure denaturation of proteins proposed earlier.     

Hydrogen-bond dynamics of water in presence of an amphiphile,tetramethylurea: signature of confinement-induced effects

Various experimental and simulation studies have suggested that the presence of amphiphilic molecules in aqueous solutions substantially perturbs the tetrahedral hydrogen-bond (H-bond) network of neat liquid water. Such structural perturbation is expected to impact H-bond lifetime of liquid water. Tetramethylurea (TMU) is an example of an amphiphile because it possesses both hydrophobic and hydrophilic moieties. Molecular dynamics simulations of (water+TMU) binary mixtures at various compositions have been performed in order to investigate the microscopic mechanism through which the amphiphiles influence the H-bond dynamics of liquid water at room temperature. Present simulations indicate lengthening of both water–water H-bond lifetime and H-bond structural relaxation time upon addition of TMU in aqueous solution. At the highest TMU mole fraction studied, H-bond lifetime and structural relaxation time are, respectively, ～4 and ～8 times longer than those in neat water. This is comparable with the slowing down of H-bond dynamics for water molecules confined in cyclodextrin cavities. Simulated relaxation profiles are multi-exponential in character at all mixture compositions, and simulated radial distribution functions suggest enhanced water–water and water–TMU interactions upon addition of TMU. No evidence for complete encapsulation of TMU by water H-bond network has been found.     

Statistical and molecular dynamics studies of buried waters in globular proteins

Buried solvent molecules are common in the core of globular proteins and contribute to structural stability. Folding necessitates the burial of polar backbone atoms in the protein core, whose hydrogen-bonding capacities should be satisfied on average. Whereas the residues in alpha-helices and beta-sheets form systematic main-chain hydrogen bonds, the residues in turns, coils and loops often contain polar atoms that fail to form intramolecular hydrogen bonds. The statistical analysis of 842 high resolution protein structures shows that well-resolved, internal water molecules preferentially reside near residues without alpha-helical and beta-sheet secondary structures. These buried waters most often form primary hydrogen bonds to main-chain atoms not involved in intramolecular hydrogen bonds, providing strong evidence that hydrating main-chain atoms is a key structural role of buried water molecules. Additionally, the average B-factor of protein atoms hydrogen-bonded to waters is smaller than that of protein atoms forming intramolecular hydrogen bonds, and the average B-factor of water molecules involved in primary hydrogen bonds with main-chain atoms is smaller than the average B-factor of water molecules involved in secondary hydrogen bonds to protein atoms that form concurrent intramolecular hydrogen bonds. To study the structural coupling between internal waters and buried polar atoms in detail we simulated the dynamics of wild-type FKBP12, in which a buried water, Wat137, forms one side-chain and multiple main-chain hydrogen bonds. We mutated E60, whose side-chain hydrogen bonds with Wat137, to Q, N, S or A, to modulate the multiplicity and geometry of hydrogen bonds to the water. Mutating E60 to a residue that is unable to form a hydrogen bond with Wat137 results in reorientation of the water molecule and leads to a structural readjustment of residues that are both near and distant to the water. We predict that the E60A mutation will result in a significantly reduced affinity of FKBP12 for its ligand FK506. The propensity of internal waters to hydrogen bond to buried polar atoms suggests that ordered water molecules may constitute fundamental structural components of proteins, particularly in regions where alpha-helical or beta-sheet secondary structure is not present.     

Role of structural water for prediction of cation binding sites in apoproteins

Structures of many metal-binding proteins are often obtained without structural cations in their apoprotein forms. Missing cation coordinates are usually updated from structural templates constructed from many holoprotein structures. Such templates usually do not include structural water, the important contributor to the ion binding energy. Structural templates are also inconvenient for taking into account structural modifications around the binding site at apo-/holo- transitions. An approach based upon statistical potentials readily takes into account structural modifications associated with binding as well as contribution of structural water molecules. Here, we construct a set of statistical potentials for Mg²⁺, Ca²⁺, and Zn²⁺ contacting with protein atoms of a different type or structural water oxygens. Each type of the cations tends to form tight contacts with protein atoms of specific types. Structural water contributes relatively more into the binding pseudo-energy of Mg²⁺ and Ca²⁺ than of Zn²⁺. We have developed PIONCA (Protein-Ion Calculator), a fast CUDA GPGPU-based algorithm that predicts ion-binding sites in apoproteins. Comparative tests demonstrate that PIONCA outperforms most of the tools based on structural templates or docking. Our software can be also used for locating bound cations in holoprotein structures with missing cation heteroatoms. PIONCA is equipped with an interactive web interface based upon JSmol.     

NMR relaxation studies on the hydrate layer of intrinsically unstructured proteins

Intrinsically unstructured/disordered proteins (IUPs) exist in a disordered and largely solvent-exposed, still functional, structural state under physiological conditions. As their function is often directly linked with structural disorder, understanding their structure-function relationship in detail is a great challenge to structural biology. In particular, their hydration and residual structure, both closely linked with their mechanism of action, require close attention. Here we demonstrate that the hydration of IUPs can be adequately approached by a technique so far unexplored with respect to IUPs, solid-state NMR relaxation measurements. This technique provides quantitative information on various features of hydrate water bound to these proteins. By freezing nonhydrate (bulk) water out, we have been able to measure free induction decays pertaining to protons of bound water from which the amount of hydrate water, its activation energy, and correlation times could be calculated. Thus, for three IUPs, the first inhibitory domain of calpastatin, microtubule-associated protein 2c, and plant dehydrin early responsive to dehydration 10, we demonstrate that they bind a significantly larger amount of water than globular proteins, whereas their suboptimal hydration and relaxation parameters are correlated with their differing modes of function. The theoretical treatment and experimental approach presented in this article may have general utility in characterizing proteins that belong to this novel structural class.     

Enhanced hydration of dipalmitoylphosphatidylcholine multibilayer by vinblastine sulphate

Vinblastine sulphate, an antimitotic and anti-inflammatory agent, modifies the thermal behaviour of the model membranes: the dipalmitoylphosphatidylcholine DPPC bilayers. The mixed DPPC and vinblastine sulphate multibilayers in the range of DPPC mole fraction 0.4 to 1 display clearly the gel-liquid crystal (chain melting) transition on the thermograms obtained with a differential scanning microcalorimeter. The molar enthalpy of this transition is slightly depressed by vinblastine sulphate (less than 10%). The temperature-composition phase diagram corresponds to a total insolubility of vinblastine sulphate inside the frozen (gel) bilayers and to a solubility of 0.2 (mole fraction) of vinblastine sulphate inside the fluid (liquid crystalline) bilayers. The dissolved vinblastine sulphate depresses the cooperativity number of the frozen ? fluid transition of the bilayers very strongly (4- to 5-times). Up to its solubility concentration, vinblastine sulphate increases the amount of the structural water of the bilayers and modifies the thermal behaviour of this water. The ‘expelled’ vinblastine sulphate molecules are retained by the polar groups of DPPC molecules and screen their electrostatic interactions with the structural water molecules. Below 0°C, the amount of the structural water, which forms the aqueous separation between two bilayers, is enhanced by vinblastine sulphate. However, the drug reduces (screens) the bilayers interaction with the structural water molecules.     

Structural modifications of air-dried tendon collagen on heating

The modification of collagen molecular packing as a function of the removal of bound and structural water have been investigated on air-dried rat tail tendon. Isothermal curves, dilatometric measurement, high and small angle X-ray diffraction patterns—recorded using conventional and synchrotron radiaiton sources respectively—have been obtained on samples heated in air at different temperatures up to 200°C. A shortening of collagen intermolecular distances and slight modifications of quaternary structure and fibre dimensions can be observed during the release of bound water. The removal of structural water is accompanied by disordering of the three polypeptide chains, a strong reduction of fibre length and d-axial spacing, and modifications of the electron density distribution inside the repeating period. The structural modifications observed during the removal of bound water and of most of the structural water, obtained on heating, are reversible. Release of the most lightly bound water, probably associated with the beginning of the depolymerization process, induces irreversible modification of the molecular packing.     

Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.     

电气石处理水对Caco-2细胞生长和碱性磷酸酶活性的影响

水是由若干水分子通过氢键结合形成的水分子团簇结构,水分子团簇的改变能产生多种生物学效应。研究了电气石对液态水团簇的影响,并以Caco-2细胞培养模型,探讨了电气石处理DMEM培养液对细胞生长和碱性磷酸酶活性的影响。研究结果显示：电气石使蒸馏水^17O核磁共振(^17O nucleaur magnetic resonance,^17O NMR)半高幅宽变窄,降低了水分子缔合度;电气石处理的DMEM培养液培养Caco-2细胞促进了细胞生长,提高了细胞碱性磷酸酶活性。结果表明电气石可降低水分子缔合度,促进细胞生长和分化。     

压力室测定根系导水率方法探讨

用压力室连续测定了玉米根系长压和降压过程的导水率,结果表明,降压过程湍 得的根系导水率显著大于用升压过程的,并且前者的相关系数大于后者,这种差异是由于这两个过程中质外体途径细胞壁空间充水量不同造成的,开始升压时,由于细胞壁空间含水量低,质外体途径阻力大,导致非结构阻力;随着压力的升高,细胞壁空间含水量增大,质外体途径导度增大,减小甚至可以消除非结构阻力,降压法可以使根系快速复水,消除传统方法因长时间复水所致根结构的改变。建议用降压法测定根系导水率。     

Hydrophobic interaction and structural changes in the solvent

The effect of structural changes in the solvent (usually water) on the thermodynamics of the hydrophobic interaction process is examined within the framework of classical statistical mechanics. The concept of the “structure of water” is first defined in a precise way, yet reflecting the conventional definition that has been, implicitly and qualitatively, employed by many authors. Using this concept, we proceed to show that structural changes in the solvent, induced by the hydrophobic interaction process, cannot affect the strength of the hydrophobic interaction. On the other hand, the entropy and enthalpy changes, associated with the same process, may well be affected. Some qualitative arguments are presented showing that large structural changes are expected from a complex solvent such as water.     

Modification of water entry (xylem vessels) and water exit (stomata) orchestrates long term drought acclimation of wheat leaves

Immediate leaf functional responses to drought, such as stomatal closure and photosynthetic rate reduction, are already known from short-term studies. We tested the hypothesis that long-term acclimation of leaves to drought includes hydraulic and stomatal anatomical changes and that gas exchange and nitrogen allocation patterns are inevitably adjusted to the new structural status. 26 structural and functional traits in one sensitive cultivar (Simeto) and two drought resistant landraces (Ntopia Heraklion 184, Kontopouli 17) of field- grown wheat (Triticum turgidum L. var. durum) were examined under four water shortage levels. Drought acclimation responses were more intense in Simeto than in the two landraces. In accordance to the working hypothesis, drought-acclimated leaves showed lower hydraulic conductance due to narrower vessels and higher stomatal and vein densities than the control leaves, resulting in a safe mode of water transfer and consumption which is essential for the survival in water limiting conditions.Irrespectively of genotype and water regime, significant correlations among structural (hydraulic characteristics, stomatal and vein patterns) and functional (gas exchange, nitrogen content) parameters were found, indicating the functional adjustment to the new structural status. The Principal Component Analysis showed that these structure-function interactions reflected the trade-off between growth and protection against water losses (Axis 1), as well as the competition between different sinks (carbon gain vs structural reinforcement and reproductive effort) in N allocation (Axis 2). Drought acclimation in wheat leaf is integrally processed by the coordination of structural and functional parameters in order to compensate for the adverse effects of water shortage. This structure??function network that regulates the transition from normal growth-mode to protection- mode, includes at least two important ??nodal points??: xylem conducting efficiency (water entry) and stomatal function (water exit). This transition also includes the redirection of nitrogen resources to different sinks.     

Structural analysis of substance P using molecular dynamics and NMR spectroscopy.

The present work is a combined structural study, using Nuclear Magnetic Resonance (NMR) and Molecular Dynamics(MD), of the amidated and the free acid forms of substance P in water and methanol. The results obtained using both approaches were compared in order to characterize the structural features of both peptides in solution. From the NMR experiments it was derived that the free acid form adopts an extended conformation at the N-terminus and a helical conformation at the C-terminal segment of the peptide in both water and methanol; these structural features are in qualitative agreement with the results of the MD simulations. No significant differences in behavior were observed between the amidated and the free acid forms of the peptide in the simulations and in the experiments carried out in water, suggesting that the different activities of these analogs are due to their different mode of interaction with the receptor rather than to their structural preferences. Finally, we propose that the structure of substance P can be partially inferred from its sequence due to the presence of a Pro-X-Pro motif on the N-terminus and a Gly-Leu sequence on the C-terminus.     

Conserved water molecules in the specificity pocket of serine proteases and the molecular mechanism of Na+ binding

Conservation of clusters of buried water molecules is a structural motif present throughout the serine protease family. Frequently, these clusters are shaped as water channels forming extensive hydrogen-bonding networks linked to the protein backbone. The most conspicuous example is the water channel present in the specificity pocket of trypsin and thrombin. In thrombin, other vitamin K-dependent proteases, and some complement factors, Na⁺ binds in this water channel and enhances allosterically the catalytic activity of the enzyme, whereas digestive and fibrinolytic proteases are devoid of such regulation. A comparative analysis of proteases with and without Na⁺ binding capability reveals the role of the water channel in maintaining the structural organization of the specificity pocket and in Na⁺ coordination. This enables the formulation of a molecular mechanism for Na⁺ binding in thrombin and leads to the identification of the structural changes necessary to engineer a functional Na⁺ site and enhanced catalytic activity in trypsin and other proteases. Proteins 30:34–42, 1998. © 1998 Wiley-Liss, Inc.     

The dynamics of protein hydration water: a quantitative comparison of molecular dynamics simulations and neutron-scattering experiments

We present results from an extensive molecular dynamics simulation study of water hydrating the protein Ribonuclease A, at a series of temperatures in cluster, crystal, and powder environments. The dynamics of protein hydration water appear to be very similar in crystal and powder environments at moderate to high hydration levels. Thus, we contend that experiments performed on powder samples are appropriate for discussing hydration water dynamics in native protein environments. Our analysis reveals that simulations performed on cluster models consisting of proteins surrounded by a finite water shell with free boundaries are not appropriate for the study of the solvent dynamics. Detailed comparison to available x-ray diffraction and inelastic neutron-scattering data shows that current generation force fields are capable of accurately reproducing the structural and dynamical observables. On the time scale of tens of picoseconds, at room temperature and high hydration, significant water translational diffusion and rotational motion occur. At low hydration, the water molecules are translationally confined but display appreciable rotational motion. Below the protein dynamical transition temperature, both translational and rotational motions of the water molecules are essentially arrested. Taken together, these results suggest that water translational motion is necessary for the structural relaxation that permits anharmonic and diffusive motions in proteins. Furthermore, it appears that the exchange of protein-water hydrogen bonds by water rotational/librational motion is not sufficient to permit protein structural relaxation. Rather, the complete exchange of protein-bound water molecules by translational displacement seems to be required.     

Numerical Simulation and Structural Optimization of the Inclined Oil/Water Separator

Improving the separation efficiency of the inclined oil/water separator, a new type of gravity separation equipment, is of great importance. In order to obtain a comprehensive understanding of the internal flow field of the separation process of oil and water within this separator, a numerical simulation based on Euler multiphase flow analysis and the realizable k-ε two equation turbulence model was executed using Fluent software. The optimal value ranges of the separator’s various structural parameters used in the numerical simulation were selected through orthogonal array experiments. A field experiment on the separator was conducted with optimized structural parameters in order to validate the reliability of the numerical simulation results. The research results indicated that the horizontal position of the dispenser, the hole number, and the diameter had significant effects on the oil/water separation efficiency, and that the longitudinal position of the dispenser and the position of the weir plate had insignificant effects on the oil/water separation efficiency. The optimal structural parameters obtained through the orthogonal array experiments resulted in an oil/water separation efficiency of up to 95%, which was 4.996% greater than that realized by the original structural parameters.