Comparative study of glutamine synthetase bound lanthanide(III) ions using NMR relaxation and lanthanide(III) luminescence techniques

Changes in the intrinsic fluorescence intensity of glutamine synthetase induced by lanthanide(III) ion binding demonstrate the existence of three types of sites for these ions. The sites are populated sequentially during titrations of the enzyme, and the first two have a stoichiometry of 1 per enzyme subunit. The number of water molecules coordinated to Eu(III) bound to the first site was determined by luminescence lifetime techniques to be 4.1 +/- 0.5. The hydration of Gd(III) bound to the same site was studied by magnetic field dependent water proton longitudinal relaxation rate measurements, and by water proton and deuteron relaxation measurements of one sample at single magnetic fields. The magnetic resonance techniques also yield a value of 4 for the hydration number.     

Hyper-mobile water is induced around actin filaments

When introduced into water, some molecules and ions (solutes) enforce the hydrogen-bonded network of neighboring water molecules that are thus restrained from thermal motions and are less mobile than those in the bulk phase (structure-making or positive hydration effect), and other solutes cause the opposite effect (structure-breaking or negative hydration effect). Using a method of microwave dielectric spectroscopy recently developed to measure the rotational mobility (dielectric relaxation frequency) of water hydrating proteins and the volume of hydration shells, the hydration of actin filament (F-actin) has been studied. The results indicate that F-actin exhibits both the structure-making and structure-breaking effects. Thus, apart from the water molecules with lowered rotational mobility that make up a typical hydration shell, there are other water molecules around the F-actin which have a much higher mobility than that of bulk water. No such dual hydration has been observed for myoglobin studied as the representative example of globular proteins which all showed qualitatively similar dielectric spectra. The volume fraction of the mobilized (hyper-mobile) water is roughly equal to that of the restrained water, which is two-thirds of the molecular volume of G-actin in size. The dielectric spectra of aqueous solutions of urea and potassium-halide salts have also been studied. The results suggest that urea and I(-) induce the hyper-mobile states of water, which is consistent with their well-known structure-breaking effect. The molecular surface of actin is rich in negative charges, which along with its filamentous structure provides a structural basis for the induction of a hyper-mobile state of water. A possible implication of the findings of the present study is discussed in relation to the chemomechanical energy transduction through interaction with myosin in the presence of ATP.     

Large-scale networks of hydration water molecules around bovine beta-trypsin revealed by cryogenic X-ray crystal structure analysis.

The hydration structure of bovine beta-trypsin was investigated in cryogenic X-ray diffraction experiments. Three crystal forms of the enzyme inhibited by benzamidine with different molecular packing were selected to deduce the hydration structure for the entire surface of the enzyme. The crystal structures in all three of the crystal forms were refined at the resolution of 1.8 A at 100 K and 293 K. The number of hydration water molecules around the enzyme at 100 K was 1.5 to two times larger than that at 293 K, indicating that the motion of hydration water was quenched by cooling. In particular, the increase in the number of hydration water molecules was prominent on flat and electrostatically neutral surface areas. The water-to-protein mass ratio and the radius of gyration of a structural model of hydrated trypsin at 100 K was consistent with the results obtained by other experimental techniques for proteins in solution. Hydration water molecules formed aggregates of various shapes and dimensions, and some of the aggregates even covered hydrophobic residues by forming oligomeric arrangements. In addition, the aggregates brought about large-scale networks of hydrogen bonds. The networks covered a large proportion of the surface of trypsin like a patchwork, and mechanically linked several secondary structures of the enzyme. By merging the hydration structures of the three crystal forms at 100 K, a distribution function of hydration water molecules was introduced to approximate the static hydration structure of trypsin in solution. The function showed that the negatively charged active site of trypsin tended to be easily exposed to bulk solvent. This result is of interest with respect to the solvent shielding effect and the recognition of a positively charged substrate by trypsin.     

Effect of progesterone on DPPC membrane: evidence for lateral phase separation and inverse action in lipid dynamics

Interactions of progesterone with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes were investigated as a function of temperature and progesterone concentration by using three non-invasive techniques namely Fourier transform infrared spectroscopy, turbidity at 440 nm, and differential scanning calorimetry. The results reveal that progesterone changes the physical properties of DPPC bilayers by decreasing the main phase-transition temperature, abolishing the pre-transition, broadening the phase-transition profile, disordering the system both in gel and liquid crystalline phase, increasing the dynamics at low concentrations whereas stabilizing the membrane at high concentrations, and inducing phase separation. Progesterone does not change the hydration of the CO groups, while it strengthens the hydrogen bonding between the PO2- groups of lipids and the water molecules around.     

Hydration effects accompanying the formation of DNA complexes with some ligands

In the range of millimeter wavelengths the dielectric properties of aqueous solutions of some biologically active ligands (potential anticarcinogen chlorophyllin; pharmacological drug caffeine; polyamine putrescine; mutagens proflavine and ethidium bromide; actinocin derivative, an analogue of antitumor antibiotic actinomycin D) and DNA complexes with these substances were studied. It was shown that complex formation is accompanied by the change in dielectric properties of the solution. These changes during interaction of DNA with the first three compounds correspond to a decrease in hydration (compared with the total hydration of free components), and in other cases they cause an increase in hydration. The number of water molecules bound with both the ligand and DNA nucleotide in the complex was estimated. The results were compared with existing models of DNA interaction with the studied substances.     

Femtosecond Hydration Map of Intrinsically Disordered α-Synuclein

Protein hydration water plays a fundamentally important role in protein folding, binding, assembly, and function. Little is known about the hydration water in intrinsically disordered proteins that challenge the conventional sequence-structure-function paradigm. Here, by combining experiments and simulations, we show the existence of dynamical heterogeneity of hydration water in an intrinsically disordered presynaptic protein, namely α-synuclein, implicated in Parkinson’s disease. We took advantage of nonoccurrence of cysteine in the sequence and incorporated a number of cysteine residues at the N-terminal segment, the central amyloidogenic nonamyloid-β component (NAC) domain, and the C-terminal end of α-synuclein. We then labeled these cysteine variants using environment-sensitive thiol-active fluorophore and monitored the solvation dynamics using femtosecond time-resolved fluorescence. The site-specific femtosecond time-resolved experiments allowed us to construct the hydration map of α-synuclein. Our results show the presence of three dynamically distinct types of water: bulk, hydration, and confined water. The amyloidogenic NAC domain contains dynamically restrained water molecules that are strikingly different from the water molecules present in the other two domains. Atomistic molecular dynamics simulations revealed longer residence times for water molecules near the NAC domain and supported our experimental observations. Additionally, our simulations allowed us to decipher the molecular origin of the dynamical heterogeneity of water in α-synuclein. These simulations captured the quasi-bound water molecules within the NAC domain originating from a complex interplay between the local chain compaction and the sequence composition. Our findings from this synergistic experimental simulation approach suggest longer trapping of interfacial water molecules near the amyloidogenic hotspot that triggers the pathological conversion into amyloids via chain sequestration, chain desolvation, and entropic liberation of ordered water molecules.     

Protein-water and protein-buffer interactions in the aqueous solution of an intrinsically unstructured plant dehydrin: NMR intensity and DSC aspects

Proton NMR intensity and differential scanning calorimetry measurements were carried out on an intrinsically unstructured late embryogenesis abundant protein, ERD10, the globular BSA, and various buffer solutions to characterize water and ion binding of proteins by this novel combination of experimental approaches. By quantifying the number of hydration water molecules, the results demonstrate the interaction between the protein and NaCl and between buffer and NaCl on a microscopic level. The findings overall provide direct evidence that the intrinsically unstructured ERD10 not only has a high hydration capacity but can also bind a large amount of charged solute ions. In accord, the dehydration stress function of this protein probably results from its simultaneous action of retaining water in the drying cells and preventing an adverse increase in ionic strength, thus countering deleterious effects such as protein denaturation.     

Dielectric Relaxation Dynamics of Water in Model Membranes Probed by Terahertz Spectroscopy

We study hydrated model membranes, consisting of stacked bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids, using terahertz time-domain spectroscopy and infrared spectroscopy. Terahertz spectroscopy enables the investigation of water dynamics, owing to its sensitivity to dielectric relaxation processes associated with water reorientation. By controlling the number of water molecules per lipid molecule in the system, we elucidate how the interplay between the model membrane and water molecules results in different water dynamics. For decreasing hydration levels, we observe the appearance of new types of water dynamics: the collective bulklike dynamics become less pronounced, whereas an increased amount of both very slowly reorienting (i.e., irrotational) and very rapidly reorienting (i.e., fast) water molecules appear. Temperature-dependent measurements reveal the interconversion between the three distinct types of water present in the system.     

Divalent paramagnetic counterions site binding in polyelectrolyte solutions. Analysis of the frequency dependence of the water protons magnetic relaxation and the characteristic parameters of "site binding"

Chemical shift and relaxation time measurements on the water protons in polyelectrolyte solutions containing divalent paramagnetic counterions have shown the existence of three types of counterions: - site bound with loss of water molecules and partial or complete release of the electrostriction in the first hydration sphere, - atmospherically trapped with no change in hydration, - free. The overall stoichiometry of the two former is in agreement with Manning's fraction of condensed counterions. A complete analysis of the frequency dependent contribution of site bound counterions to the water protons relaxation times leads us to interesting conclusions on the modifications of the first hydration shell and on the life time of site binding.     

Direct imaging of individual intrinsic hydration layers on lipid bilayers at Angstrom resolution

The interactions between water and biological molecules have the potential to influence the structure, dynamics, and function of biological systems, hence the importance of revealing the nature of these interactions in relation to the local biochemical environment. We have investigated the structuring of water at the interface of supported dipalmitoylphosphatidylcholine bilayers in the gel phase in phosphate buffer solution using frequency modulation atomic force microscopy (FM-AFM). We present experimental results supporting the existence of intrinsic (i.e., surface-induced) hydration layers adjacent to the bilayer. The force versus distance curves measured between the bilayer and the AFM tip show oscillatory force profiles with a peak spacing of 0.28 nm, indicative of the existence of up to two hydration layers next to the membrane surface. These oscillatory force profiles reveal the molecular-scale origin of the hydration force that has been observed between two apposing lipid bilayers. Furthermore, FM-AFM imaging at the water/lipid interface visualizes individual hydration layers in three dimensions, with molecular-scale corrugations corresponding to the lipid headgroups. The results demonstrate that the intrinsic hydration layers are stable enough to present multiple energy barriers to approaching nanoscale objects, such as proteins and solvated ions, and are expected to affect membrane permeability and transport.     

Effects of metal ion and solute conformation change on hydration of small amino acid

The effects of metal ion and solute conformation change on the structures, energetic and dynamics of water molecules in the first hydration shell of amino acid were studied, using three forms of alanine (Ala) and Li(+)/Ala as model molecules. The theoretical investigations were started with construction of the test-particle model (T-model) potentials for all molecules involved and followed by molecular dynamics (MD) simulations of [Ala](aq) and [Li(+)/Ala](aq) at 298 K. The MD results showed that the hydrogen bond (H-bond) networks of water at the functional groups of Ala are strengthened by the metal ion binding, whereas the rotation of the N-C(alpha) bond from the angle phi=0 degrees to 180 degrees brings about smaller effects which cannot be generalized. It was also shown that the dynamics of water molecule in the first hydration shell of amino acid could be estimated from the total-average potential energy landscapes and the water exchange diagrams. The MD results suggested inclusion of an additional dynamic step in the water exchange process, in which water molecule moves inside a channel within the first hydration shell of solute, before leaving the channel at some point. The theoretical results reported in the present work iterated the necessity to include explicit water molecules in the model calculations.     

Structural principles of B-DNA grooves hydration in fibers as revealed by Monte Carlo simulations and X-ray diffraction

Being interested in possible effects of sequence-dependent hydration of B-DNA with mixed sequence in fibers, we performed a series of Monte Carlo calculations of hydration of polydeoxyribonucleotides in B form, considering all sequences with dinucleotide repeat. The computational results allow the ten base-stacking types to be classified in accordance with their primary hydration in the minor groove. As a rule, the minor groove is occupied by two water molecules per base pair in the depth of the groove, which are located nearly midway between the planes of successive base pairs and symmetrically according to the dyad there. The primary hydration of the major groove depends on the type of the given base pair. The coordinates of 3 water molecules per base pair in the depth of the major groove are determined by the type of this pair together with its position and orientation in the helix, and are practically independent on the adjacent base pairs. A/T-homopolymer tracts do not fit into this hydration pattern; the base pair edges are hydrated autonomously in both grooves. Analysis of the Li-B-DNA x-ray diffraction intensities reveals those two water positions in the minor groove. In the major groove, no electronic density peaks in sufficient distance from the base edges were found, thus confirming the absence of any helical invariance of primary hydration in this region. With the help of the rules proposed in this paper it is possible to position the water molecules of the first hydration shell in the grooves of canonical B-DNA for any given sequence.     

Hydration in proteins observed by high-resolution neutron crystallography

It is well known that water molecules surrounding a protein play important roles in maintaining its structural stability. Water molecules are known to participate in several physiological processes through the formation of hydrogen bonds. However, the hydration structures of most proteins are not known well at an atomic level at present because X-ray protein crystallography has difficulties to localize hydrogen atoms. In contrast, neutron crystallography has no problem in determining the position of hydrogens with high accuracy.1 In this article, the hydration structures of three proteins are described- myoglobin, wild-type rubredoxin, and a mutant rubredoxin-the structures of which were solved at 1.5- or 1.6-A resolution by neutron structure determination. These hydration patterns show fascinating features and the water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen bond formation and dynamics of hydration. Our results further show that there are strong relationships between these shapes and the water environments.     

Hydration of RNA base pairs

The hydration patterns around the RNA Watson-Crick and non-Watson-Crick base pairs in crystals are analyzed and described. The results indicate that (i) the base pair hydration is mostly "in-plane"; (ii) eight hydration sites surround the Watson-Crick G-C and A-U base pairs, with five in the deep and three in the shallow groove, an observation which extends the characteristic isostericity of Watson-Crick pairs; (iii) while the hydration around G-C base pairs is well defined, the hydration around A-U base pairs is more diffuse; (iv) the hydration sites close to the phosphate groups are the best defined and the most recurrent ones; (v) a string of water molecules links the two shallow groove 2'-hydroxyl groups, and (vi) the water molecules fit into notches, the size and accessibility of which are almost as important as the number and strength of the hydrophilic groups lining the cavity. Residence times of water molecules at specific hydration sites, inferred from molecular dynamics simulations, are discussed in the light of present data.     

Assessment of thermal dehydration using the human eye: What is the potential?

Human hydration assessment is a key component for the prevention and proper treatment of heat-related fluid and electrolyte imbalances within military, sports and clinical medicine communities. Despite the availability of many different methods for assessing hydration status, the need for a valid method or technology that is simple, rapid, non-invasive, universal (detects both hypertonic and isotonic hypovolaemia) and is applicable for static (single point in time) and dynamic (change across time) hydration assessment is widely acknowledged. The eye is one candidate body region that might afford such a measure given the intricate balance between ocular dynamics (tear and aqueous humor formation) and blood (plasma osmolality and volume), which is considered the criterion measure for hydration assessment. The aim of this review is to introduce and discuss the potential for using ocular measurements for non-invasive hydration assessment, including tear fluid osmolarity (Tosm), non-invasive tear break-up time (NITBUT) and intraocular pressure (IOP). There is a relevant physiological basis for testing the merit of ocular measures for human hydration assessment and recent data indicate that Tosm and IOP may have utility. Further investigations are warranted to determine the degree to which ocular measures can act as accurate and reliable non-invasive hydration status markers.     

Effects of water gradients and use of urea on skin ultrastructure evaluated by confocal Raman microspectroscopy

The rather thin outermost layer of the mammalian skin, stratum corneum (SC), is a complex biomembrane which separates the water rich inside of the body from the dry outside. The skin surface can be exposed to rather extreme variations in ambient conditions (e.g. water activity, temperature and pH), with potential effects on the barrier function. Increased understanding of how the barrier is affected by such changes is highly relevant for regulation of transdermal uptake of exogenous chemicals. In the present study we investigate the effect of hydration and the use of a well-known humectant, urea, on skin barrier ultrastructure by means of confocal Raman microspectroscopy. We also perform dynamic vapor sorption (DVS) microbalance measurements to examine the water uptake capacity of SC pretreated with urea. Based on novel Raman images, constructed from 2D spectral maps, we can distinguish large water inclusions within the skin membrane exceeding the size of fully hydrated corneocytes. We show that these inclusions contain water with spectral properties similar to that of bulk water. The results furthermore show that the ambient water activity has an important impact on the formation of these water inclusions as well as on the hydration profile across the membrane. Urea significantly increases the water uptake when present in skin, as compared to skin without urea, and it promotes formation of larger water inclusions in the tissue. The results confirm that urea can be used as a humectant to increase skin hydration.     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 μg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 μm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 μg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

微波热声成像技术用于人体甲状腺检测

本文首次通过人体实验验证了微波热声成像技术用于人体甲状腺检测的可行性.文章首先讨论了该技术用于人体甲状腺检测的可行性;然后对3名志愿者的健康甲状腺进行了微波热声成像实验.结果表明:微波热声成像能够对人体健康甲状腺进行清晰成像,能够真实反映皮肤、甲状腺和气管等不同组织的结构特征;并且一次完整的检测过程时间约5 s,系统操作简单成像快速.综上所述,微波热声成像技术有望为甲状腺疾病的基础研究和临床诊断提供一种新的影像学参考.     

Hydration properties of DNA-lysine gels by microwave dielectric measurements as a function of temperature

Dielectric measurements by a cavity perturbation method at 10 GHz in the temperature range from-20°C to +45°C are reported for aqueous gels of herring sperm DNA in the presence of 1 or 3 lysine molecules per nucleotide. Measurements for lysine-water and DNA-water systems are also reported. The experimental results can be accounted for by the presence of interfacial water, with dielectric properties different from those of bulk water, and are analyzed in terms of a three component equation (solute molecules, interfacial water and bulk water) to calculate hydration parameters of the systems. The lysine molecule is found to coordinate a particular number of water molecules, in agreement with the literature. The specific hydration of DNA is reduced by the presence of lysine, indicating a direct interaction between the polyion and the aminoacid: a decrease to about 50% was observed at a ratio of one molecule of lysine per nucleotide. A suggestion is made that the interaction is mainly electrostatic in nature.     

Sequence Dependencies of DNA Deformability and Hydration in the Minor Groove

DNA deformability and hydration are both sequence-dependent and are essential in specific DNA sequence recognition by proteins. However, the relationship between the two is not well understood. Here, systematic molecular dynamics simulations of 136 DNA sequences that differ from each other in their central tetramer revealed that sequence dependence of hydration is clearly correlated with that of deformability. We show that this correlation can be illustrated by four typical cases. Most rigid basepair steps are highly likely to form an ordered hydration pattern composed of one water molecule forming a bridge between the bases of distinct strands, but a few exceptions favor another ordered hydration composed of two water molecules forming such a bridge. Steps with medium deformability can display both of these hydration patterns with frequent transition. Highly flexible steps do not have any stable hydration pattern. A detailed picture of this correlation demonstrates that motions of hydration water molecules and DNA bases are tightly coupled with each other at the atomic level. These results contribute to our understanding of the entropic contribution from water molecules in protein or drug binding and could be applied for the purpose of predicting binding sites.