Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.     

Thermostability of DNA and its connection with the glassing process

Using differential scanning calorimetry, the thermal denaturation of calf thymus DNA with different content of water (from 12 to 92%) was investigated. Dependences of melting temperature and enthalpy on the biopolymer hydration degree were established. Within the range of water concentrations from 92 to 50% the values of thermodynamic parameters of denaturation were obtained being in good agreement with the published data. Besides, a calorimetric manifestation of renaturation process at different cooling conditions after denaturation was studied. Special attention was paid to thermal properties of denatured and native DNA in the samples containing only the bound water. The temperature dependence of heat capacity in the denatured samples, which have completely lost their renaturation ability due to the proper thermal treatment, demonstrated a characteristic jump of thermal capacity. The value of this jump has been determined to be equal to 1.0 cal/g. degree C, related to dry weight, and almost not dependent on humidity. Temperature position of the jump (Tg) depends on the content of water which serves as a plasticizer. It is shown that the observed anomaly demonstrates all the properties characteristic of vitrification process in synthetic polymers and proteins. General similarity of thermal properties of the samples of native DNA, containing only the bound water, with those of denatured DNA also indicates a transition from the glassy into the rabber-like state. A possibility of existence of both native and denatured DNA in the glassy state at room temperature for the samples with low humidity (about 25%) has been demonstrated experimentally. It can be suggested that the formation of glassy state at dehydration of native DNA ensures its thermostability and the ability of restoration of its functional properties at a subsequent dehydration.     

The effect of hydrogen ions on the B-A transition in DNA

The effects of hydrogen ions binding to DNA on its secondary structure and B to A transition were studied by methods of X-ray diffraction and infrared spectroscopy. Helical parameters of DNA molecules with different degrees of protonation were determined. It was shown that H+-ions binding stabilize the B form of DNA in fibers in the wide range of water and inorganic salt content. Only 0.03 H+-ions bound to each nucleotide are sufficient to prevent B to A transition caused by a relative humidity decrease in DNA fibers, containing 4% of NaCl. The effective stabilization of the DNA B form by H+-ions binding is explained by modifications in DNA - solvent molecules interactions, especially in the major groove of double helices.     

Infrared and X-ray diffraction study of the effect of protonation of DNA on its B-to-A transition

The influence of H+ on the secondary structure of DNA and on its B-to-A transition has been studied by employing X-ray diffraction and infrared spectroscopy. Helical parameters for DNA molecules with different degrees of protonation were determined. It was shown that H+ binding stabilizes the B-form of DNA in fibers over a wide range of water and inorganic salt content. Only 0.03 H+ bound per nucleotide is sufficient to prevent the B-to-A transition caused by decreasing relative humidity in DNA fibers containing 4% NaCl. The effectiveness of B-form stabilization by H+ is explained by changes in DNA-solvent molecule interactions, especially in the major groove of double helices.     

影响蝴蝶兰类原球茎耐脱水性的主要因素

为探讨蝴蝶兰(Phalaenopsis spp.)类原球茎(protocorm-like body,PLB)耐脱水性的主要影响因素,对PLB的平均粒重、含水率、脱水相对湿度、时间、温度、光周期与耐脱水性的关系进行了研究.结果表明,PLB的平均粒重与脱水后失水率、含水率、相对电导率、成活率呈显著或极显著相关.在较高湿度下...     

Dehydrating phospholipid vesicles measured in real-time using ATR Fourier transform infrared spectroscopy

According to the water replacement hypothesis, trehalose stabilizes dry membranes by preventing the decrease in spacing between adjacent phopspholipid headgroups during dehydration. Alternatively, the water-entrapment hypothesis postulates that in the dried state sugars trap residual water at the biomolecule sugar interface. In this study, Fourier transform infrared spectroscopy with an attenuated total reflection accessory was used to investigate the influence of trehalose on the dehydration kinetics and residual water content of egg phosphatidylcholine liposomes in real time under controlled relative humidity conditions. In the absence of trehalose, the lipids displayed a transition to a more ordered gel phase upon drying. The membrane conformational disorder in the dried state was found to decrease with decreasing relative humidity. Even at a relative humidity as high as 94% the conformational disorder of the lipid acyl chains decreased after evaporation of the bulk water. The presence of trehalose affects the rate of water removal from the system and the lipid phase behavior. The rate of water removal is decreased and the residual water content is higher, as compared to drying in the absence of trehalose. During drying, the level of hydrogen bonding to the head groups remains constant. In addition, the conformational disorder of the lipid acyl chains in the dried state more closely resembles that of the lipids in the fully hydrated state. We conclude that water entrapment rather than water replacement explains the effect of trehalose on lipid phase behavior of phosphatidylcholine lipid bilayers during the initial phase of drying.     

Infrared linear dichroism studies of DNA-drug complexes: quantitative determination of the drug-induced restriction of the B-A transition.

The B-A transition of films or fibers of NaDNA occurs at a relative humidity of 75-85%. The fraction of DNA that changed the conformation from B to A form can be determined quantitatively by infrared linear dichroism. DNA-binding drugs can 'freeze' a fraction of DNA in the B form. This fraction of DNA is in the B form and cannot be converted to A-DNA even at a reduced relative humidity of 54%. The 'freezing' potentiality of various drugs can be described by the 'freezing' index, FI, expressed in base pairs per added drug. Drugs with a high value of FI (more than eight base pairs per drug) were observed among both intercalating and groove-binding drugs. High values of FI imply restriction of the conformational flexibility of DNA significantly going beyond the binding site of the drug. This long-range effect of drugs on the conformational flexibility of DNA may be connected with the molecular mechanism of drug action. The freezing index FI is a new quantitative parameter of drug-DNA interaction that should be considered as a valuable tool for drug design.     

Spin-lattice Relaxation in the Proton NMR of Hydrated Tobacco Cut-fillers

The spin-lattice relaxation time was measured by proton NMR of hydrated tobacco cut-fillers. The relaxation decays of adsorbed water were expressed by a single phase system below 70% relative humidity, while a two-phase system was applicable to water adsorbed at more than 80% relative humidity. From the two-phase model, it was considered that 0.12–0.13 kg water/kg dry tobacco is bound water.     

Modeling high-resolution hydration patterns in correlation with DNA sequence and conformation

Hydration around the DNA fragment d(C5T5).(A5G5) is presented from two molecular dynamics simulations of 10 and 12 ns total simulation time. The DNA has been simulated as a flexible molecule with both the CHARMM and AMBER force fields in explicit solvent including counterions and 0.8 M additional NaCl salt. From the previous analysis of the DNA structure B-DNA conformations were found with the AMBER force-field and A-DNA conformations with CHARMM parameters. High-resolution hydration patterns are compared between the two conformations and between C.G and T.A base-pairs from the homopolymeric parts of the simulated sequence. Crystallographic results from a statistical analysis of hydration sites around DNA crystal structures compare very well with the simulation results. Differences between the crystal sites and our data are explained by variations in conformation, sequence, and limitations in the resolution of water sites by crystal diffraction. Hydration layers are defined from radial distribution functions and compared with experimental results. Excellent agreement is found when the measured experimental quantities are compared with the equivalent distribution of water molecules in the first hydration shell. The number of water molecules bound to DNA was found smaller around T.A base-pairs and around A-DNA as compared to B-DNA. This is partially offset by a larger number of water molecules in hydrophobic contact with DNA around T.A base-pairs and around A-DNA. The numbers of water molecules in minor and major grooves have been correlated with helical roll, twist, and inclination angles. The data more fully explain the observed B-->A transition at low humidity.     

The B- to A-DNA transition and the reorganization of solvent at the DNA surface

DNA geometry depends on relative humidity. Using the CHARMM22 force field to push B-DNA to A-DNA, a molecular dynamics simulation of a mixed-sequence 24-basepair DNA double-stranded oligomer, starting from B-DNA, was carried out to explore both the mechanism of the transition and the evolution of hydration patterns on the surface of DNA. Over the 11-ns trajectory, the transition recapitulates the slide-first, roll-later mechanism, is opposed by DNA electrostatics, and is favored by an increasing amount of condensed sodium ions. Hydration was characterized by counting the hydrogen bonds between water and DNA, and by the number of water bridges linking two DNA atoms. The number of hydrogen bonds between water and DNA remains constant during the transition, but there is a 40% increase in the number of water bridges, in agreement with the principle of economy of hydration. Water bridges emerge as delicate sensors of both structure and dynamics of DNA. Both local flexibility and the frustration of the water network on the surface of DNA probably account for the low populations and short residence times of the bridges, and for the lubricant role of water in ligand-DNA interactions.     

The effect of structural water molecules on the normal mode spectrum of dTn . dAn x dTn DNA

In this paper we present a theoretical treatment of triplex B type DNA hydration using normal mode calculation techniques. Discrete solvent is added as spines of hydration in the Watson-Crick and Crick-Hoogsteen grooves as well as water bridges between the Phosphate groups. The effect of binding the discrete structural waters on the normal mode of vibration of the system was studied by introducing a parameter, Xw, that is proportional to the degree of water binding and inversely proportional to the relative humidity (RH) of the system. We examined the variation of the dipole moments of characteristic modes with Xw. The results show that there is a direct relationship between the degree of binding of the water molecules to the atoms in the triple helix, the relative humidity of the system and the conformation and stability of the triple helix. At high RH and Xw = 0:0 the triple helix has mostly B type conformation characteristics, with C'2 -endo sugars. The emergence of normal modes of vibration characteristic to the A type conformation (C'3 - endo sugars) at Xw = 0:4 and 60% RH indicates a conformational shift towards A-type for some of the sugars between Xw = 0.2 (80% RH) and Xw = 0.4 (60% RH). These results are in agreement with the "economy of hydration hypothesis" of Saenger (Saenger et al., 1986) which maintains that the main difference in the hydration of A- and B- forms of DNA is the presence of water bridges between adjacent Phosphate groups in the low-hydration A-form but not in the B- form. Free energy calculations for the triplex DNA with structural waters show that there is a minimum of the free energy at Xw = 0.2 and the free energy increases with Xw and becomes larger than the free energy of the B conformation without structural waters for Xw equal to and larger than 0.4. This result indicates that the B conformation is more stable with bound structural water molecules (for degrees of water binding that are not over 20% higher than the degree of binding between bulk water molecules). The structural water molecules are bound much tighter in the A conformation than in the B conformation. The model predicts that the B to A transition occurs at higher relative humidities in D2O than in H2O. Part of these results (Dadarlat, 1997) have been subsequently confirmed by the experimental work and MD simulations of Ouali (Ouali et al., 1997). The experimental results showed that the N-type sugars corresponding to the A conformation are clearly detected below 75% RH.     

The origin of the A to B transition in DNA fibers and films

We have studied the hydration of Na-DNA and Li-DNA fibers and films, measuring water contents, x-ray fiber diffraction patterns, low-frequency Raman spectra (below 100 cm^?1), high-frequency Raman spectra (600–1000 cm^?1), and swelling, as a function of relative humidity. Most samples gain weight equilibrium (though not conformational equilibrium) in one day. The volume occupied by a base pair as the DNA is hydrated (obtained from the x-ray and swelling data) shows anomalies for the case of Na-DNA in the region where the A-form occurs. Our Raman and x-ray data reproduce the well-known features of the established conformational transitions, but we find evidence in the Raman spectra and optical properties of a transition to what may be a disordered B-like conformation in Na-DNA below 40% relative humidity. We have studied the effects of crystallinity on the A to B transition. We find that the transition to the B-form is impeded in highly crystalline samples. In most samples, the transition occurs in three days (after putting the sample at 92% relative humidity) but in highly crystalline samples, the transition may take months. By comparing the high-frequency Raman spectra of highly ordered and disordered films, we show that the extent of crystallinity controls the amount of A-DNA formed when ethanol is used to dehydrate the films. We show that rapid dehydration (by laser heating) does not result in a B to A transition. A fiber that gives A-type x-ray reflections probably contains B-like material in noncrystalline regions. The low-frequency Raman spectrum is dominated by a band at about 25 cm^?1 in both Na- and Li-DNA. Another band is seen near 35 cm^?1 in Na-DNA at humidities where the sample is in the A-form. In contrast to earlier reports, we find that the Raman intensity does not depend on fiber orientation relative to the scattering vector. The “35-cm^?1” band is largely depolarized (i.e. vertical polarization incident and horizontal polarization scattered, VH, or vice versa, HV) while the “25-cm^?1” band appears in both VV, VH and HV polarizations. These bands are all weaker in HH polarization. The “25-cm^?1” band may be due to a shearing motion of the phosphates and their associated counterions, while the “35-cm^?1” band may be characteristic of A-DNA crystallites. We consider mass-loading, relaxational coupling to the hydration shell, and softening of interatomic potentials as possible explanations of the observed softening of the low-frequency Raman bands on hydration. Relaxation data suggest that the added water binds tightly (on these time scales) and a mass-loading model accounts for the observed softening rather well. We conclude that the A to B transition is not driven by softening of the “25-cm^?1” band. Rather, it is most probably a consequence of crystal-packing forces, with the more regular A-form favored in crystals when these forces are strong.     

Evidence of a maternal effect that protects against water stress in larvae of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

We report that the ability to absorb water vapor from the air in larvae of the American dog tick, Dermacentor variabilis, changes depending upon moisture conditions where the eggs develop. When development occurs at lower relative humidities, resultant larvae can replenish water stores, maintain water balance, and survive at relative humidities as low as 75-85% RH, a range that agrees with previously published values for the critical equilibrium humidity or CEH. In contrast, exposure to high relative humidity conditions during development elevates the CEH to 93-97% RH. These larvae can survive only at relative humidities that are close to saturation, as 93% RH is a dehydrating atmosphere. For these larvae, absorption at 97% RH can be prevented by blocking the mouthparts with wax, indicating that an upward shift has occurred in the moisture threshold where the active mechanism for water vapor absorption operates. Based on transfer experiments between low and high relative humidities, the CEH of larvae is determined by the relative humidity experienced by the mother rather than the moisture conditions encountered by eggs after they are laid. The fact that no changes in body water content, dehydration tolerance limit and water loss rate were observed implies that adjustments to the CEH conferred by the mother have the adaptive significance of enabling larvae to maintain water balance by limiting the range of hydrating atmospheres.     

Water molecules and exchangeable hydrogen ions at the active centre of bacteriorhodopsin localized by neutron diffraction. Elements of the proton pathway?

Neutron diffraction is used to localize water molecules and/or exchangeable hydrogen ions in the purple membrane by H2O/2H2O exchange experiments at different values of relative humidity. At 100% relative humidity, differences in the hydration between protein and lipid areas are observed, accounting for an excess amount of about 100 molecules of water in the lipid domains per unit cell. A pronounced isotope effect was observed, reproducibly showing an increase in the lamellar spacing from 60 A in 2H2O to 68 A in H2O. At 15% relative humidity, the positions of exchangeable protons became visible. A dominant difference density peak corresponding to 11 +/- 2 exchangeable protons was detected in the central part of the projected structure of bacteriorhodopsin at the Schiff's base end of the chromophore. A difference density map obtained from data on purple membrane films at 15% relative humidity in 2H2O, and the same sample after complete drying in vacuum, revealed that about eight of these protons belong to four water molecules. This is direct evidence for tightly bound water molecules close to the chromophore binding site of bacteriorhodopsin, which could participate in the active steps of H+ translocation as well as in the proton pathway across this membrane protein.     

Changes in the water binding characteristics of the cell walls from transgenic Nicotiana tabacum leaves with enhanced levels of peroxidase activity

Pore size in the cell wall matrix may affect cell wall–water relations, particularly under osmotic stress. Cross linkage of plant cell wall matrix polymers is an important step in the formation of this structure and peroxidases have been proposed to catalyse the cross-linking of phenolic constituents. Transgenic tobacco ( Nicotiana tabacum ) plants expressing a basic tomato peroxidase gene (TPX2) showed increased apoplastic ferulic acid peroxidase activity in mature leaves. This enhanced activity was not associated with a decreased leaf growth. Differential scanning calorimetry (DSC) of control dried cell walls showed a putative glass transition, after Ca²⁺ removal, that was absent in the transgenic line. This would indicate that transgenic walls were more rigid. DSC analysis of water-hydrated cell wall preparations distinguished two pools of water, freezable and non-freezable water. The amount of non-freezable water, which corresponds to strongly bound water, was higher in the transgenic line (64 versus 55%). DSC thermograms of the transgenic cell wall were displaced to lower temperatures, and this may be interpreted as the result of a stronger interaction between this freezable water and this wall. Water sorption and desorption isotherms, obtained at relative humidity ranging from 5 to 93%, demonstrated the presence of very strongly bound water in the transgenic cell walls that was absent in controls. Water sorption–desorption hysteresis of the isotherms was evident in the control wall but not in the transgenic line. These changes in cell wall–water interaction seem to be relevant at the organ level because leaf discs of transgenic plants maintained higher relative water content than control discs, at water potentials between −1.05 and−2.31 MPa.     

Solid-vapor interactions: Influence of environmental conditions on the dehydration of carbamazepine dihydrate

The goal of this research was a phenomenological study of the effect of environmental factors on the dehydration behavior of carbamazepine dihydrate. Dehydration experiments were performed in an automated vapor sorption apparatus under a variety of conditions, and weight loss was monitored as a function of time. In addition to lattice water, carbamazepine dihydrate contained a significant amount of physically bound water. Based on the kinetics of water loss, it was possible to differentiate between the removal of physically bound water and the lattice water. The activation energy for the 2 processes was 44 and 88 kJ/mol, respectively. As expected, the dehydration rate of carbamazepine dihydrate decreased with an increase in water vapor pressure. While dehydration at 0% relative humidity (RH) resulted in an amorphous anhydrate, the crystallinity of the anhydrate increased as a function of the RH of dehydration. A method was developed for in situ crystallinity determination of the anhydrate formed. Dehydration in the presence of the ethanol vapor was a 2-step process, and the fraction dehydrated at each step was a function of the ethanol vapor pressure. We hypothesize the formation of an intermediate lower hydrate phase with unknown water stoichiometry. An increase in the ethanol vapor pressure first led to a decrease in the dehydration rate followed by an increase. In summary, the dehydration behavior of carbamazepine dihydrate was evaluated at different vapor pressures of water and ethanol. Using the water sorption apparatus, it was possible to (1) differentiate between the removal of physically bound and lattice water, and (2) develop a method for quantifying, in situ, the crystallinity of the product (anhydrate) phase.     

Group influence on water conservation in the giant Madagascar hissing-cockroach, Gromphadorhina portentosa (Dictyoptera: Blaberidae)

Abstract. . Under laboratory conditions the hissing-cockroach Gromphadorhina portentosa (Schaum) forms clusters, which appears to be an adaptive behaviour to help reduce water loss. Adults grouped together retain water nearly twice as effectively as isolated individuals. This 'group effect' complements the cockroach's large body size (small surface area to volume ratio) to lower the rate of water loss still further. Despite a modest 28% tolerance limit for weight loss during dehydration, adult females survive absolute drying conditions of 0 % relative humidity without food and free water for at least a month, showing their impressive capacity for water retention. Rates of water loss of immature adults correlate with size, and no transition temperature is detected in females. To replenish water stores, cockroaches drank liquid water; there is no evidence for water gain by water vapour absorption. The profound impact exerted by the 'group' for water conservation suggests that members of this species live huddled together in nature, particularly during the long tropical dry season in order to conserve water, and this adds to previous evidence for the existence of a probable social structure.     

Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L^?1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ～25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.     

Influence of moisture on the crystal forms of niclosamide obtained from acetone and ethyl acetate

The purpose of this study was to elucidate the formation of crystal hydrates of niclosamide and to delineate the effect of relative humidity on the crystal forms obtained from acetone and ethyl acetate. Recrystallization of niclosamide was performed in the presence and absence of moisture. Two hydrates and their corresponding anhydrates were isolated. The hydrates obtained by the process of recrystallization from acetone (Form I) and that obtained from ethyl acetate (Form II) were classified based on differences in their dehydration profile, crystal structure, shape, and morphology. Crystals obtained in the absence of moisture were unstable, and when exposed to the laboratory atmosphere transformed to their corresponding hydrates. Differential scanning calorimetry thermograms indicate that Form I changes to an anhydrate at temperatures below 100°C, while Form II dehydrates in a stepwise manner above, 140°C. This finding was further confirmed by thermogravimetric analysis. Dehydration of Form II was accompanied by a loss of structural integrity, demonstrating that water molecules play an important role in maintaining its crystal structure. Form I, Form II, and the anhydrate of Form II showed no significant moisture sorption over the entire range of relative humidity. Although the anhydrate of Form I did not show any moisture uptake at low humidity, it converted to the monohydrate at elevated relative humidity (>95%). All forms could be interconverted depending on the solvent and humidity conditions.     

Preliminary x-ray studies of the interaction of salmon sperm DNA with spermine

The interaction of spermine with salmon sperm DNA was studied by X-ray diffraction methods. In the fibers of the complex, DNA was in the B-configuration with ten nucleotide pairs per turn (34 Å) of the helix at 92% or higher relative humidity, while, at 75% and lower relative humidity, it was at least partly in the A-configuration, which was not observed in a similar experiment by Suwalsky et al. (1969) with calf thymus DNA. The A to B transition of the complex fibers was reversible.