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A differential scanning micro-calorimetric (DSC) investigation has been performed to study the thermal stability of lysozyme solvated in glycerol as a function of the water content h (grams of water/grams of lysozyme). The unfolding process is strongly dependent on h, as it is witnessed by the behavior of the melting temperature Tm which shows a significant drop in the hydration range from 0 h to 0.4/0.5 h. The specific heat difference ΔCp between denatured and native state also displays an important variation for water amounts lower than ca. 0.5 h. For higher water amounts, both Tm and ΔCp seem to attain constant values. Such hydration dependent behavior is reminiscent of the trend exhibited by some quantities characterizing the protein internal dynamics on the pico- and nano-second timescale, as estimated by elastic neutron scattering. Also the protein mobility, which is measured through mean square displacements (MSD), increases mainly for water amounts lower than ca. 0.5 h, in the same range where the protein molecular rigidity rapidly drops down. This behaviour emphasizes the crucial role played by hydration water in determining both the protein thermodynamic and dynamic quantities.

The hydration properties of two biologically relevant molecules, trimethylamine-N-oxide (TMAO) and tert-butyl alcohol (TBA), were investigated by monitoring the effects of these two solutes on the near-infrared (NIR) spectra of water. In particular, the 1450-nm ν1 + ν3 water combination band (ν1 is the symmetric stretching and ν3 is the asymmetric stretching) and the 1928-nm ν2 + ν3 band (ν2 is the bending) were recorded at 25 °C in aqueous solutions of TBA and TMAO over the 0−0.1 and 0−0.05 solute mole fraction intervals for TBA and TMAO, respectively. NIR data show, in agreement with molecular dynamics simulations and other suggestions found in the literature, that on the whole water molecules are more tightly coordinated by TMAO than by TBA. Furthermore, nonadditive perturbations of the water's H-bond network are observed for TBA and are absent in the TMAO case. These results are discussed in connection to the significantly different action exerted by these two solutes on typical processes governed by ...

We studied the overall atomic mobility of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles in deuterated cyclohexane $({\mathrm{C}}_{6}{\mathrm{D}}_{12})$ as a function of the molar ratio $W=[{\mathrm{D}}_{2}\mathrm{O}]/[\mathrm{AOT}]$ with an incoherent quasielastic neutron scattering experiment at high energy resolution. For the almost anhydrous sample, the quasielastic broadening can be entirely attributed to the reverse micelle global motion, by considering explicitly both the rotational and the translational terms. As W increases above a threshold value $W\ensuremath{\sim}1$ a wide quasielastic signal appears, which has been interpreted as the onset of a hydration-dependent intrinsic micelle dynamics. Such a contribution, which involves the AOT monomer hydrogen atoms, has a characteristic time of 0.2 ns. This result has been compared with previous dielectric measurements, which detected a relaxation process of the AOT fully hydrated head groups with the same characteristic time. The internal macromolecule mobility evaluated as a function of W numerically correlates with that of the mobile head groups, calculated by dielectric measurements. These findings suggest that both the hydrophobic and hydrophilic moieties dynamics is activated by the progressive hydration of the reverse micelle.

Dodecyl-dimethyl-amine-oxide (DDAO) is an amphiphile that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Thus it realizes ionic or non-ionic micelles of easily controlled composition. These micelles, partially charged, realize an efficient support on which DNA can be linked in a controlled manner. In this Letter we report a study on DNA/DDAO complex formation using two physical techniques, dielectric spectroscopy (DS) and circular dichroism (CD). At pH 8, where the micelle is in a neutral state, no formation of a complex DNA/DDAO is evident. At pH 7, where the degree of ionization of the micelle is very low, the formation of the complex has been observed by the two techniques. Coherent results on the formation of the complex as a function of DDAO concentration and its influence on DNA conformation are presented.

The dielectric properties of dodecyldimethylamine oxide [DDAO] in salt-free aqueous solutions under different pH conditions have been measured in the frequency range from 400 kHz to 100 MHz. DDAO is an amphyphile that may exist either in a neutral or a cationic protonated form, depending on the pH of the aqueous solution. This condition realizes, above the cmc, ionic or nonionic micelles in an easily controlled way. While the dielectric response of a neutral micelle, in the low-concentration limit, is rather flat, a dielectric dispersion occurs at pH values lower than pH = 7. This dielectric dispersion is attributed to an orientational polarization mechanism of the entire micelle, in addition to the usual Maxwell−Wagner effect. This orientational polarization is due to an electric dipole moment induced by protonation of the micellar surface. The average size of the micelle, considered as a rigid sphere of radius RH, has been evaluated from the relaxation time of the observed dielectric relaxation. We foun...

We report results of a detailed dielectric, IR and elastic neutron scattering study of bis(2-ethylhexyl)sulfosuccinate (AOT) based micellar systems in CCl4 as a function of the molar ratio W=[H2O]/[AOT]. The aim is to explore the connection between hydration and dynamical properties of the reverse AOT micelles. We restricted our analysis to the region of small amounts of water added (0

We performed dielectric measurements on sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles in CCl4 as a function of the molar ratio W=[D2O]/[AOT], for a small amount of deuterated water (0.2

In this paper, we report a study of the effect of solvent viscosity on both translational and rotational dynamics of a simple model protein: the egg white lysozyme. For this, we investigated the dynamical properties of lysozyme in mixtures water–glycerol by means of parallel measurements of photon correlation spectroscopy (PCS) and dielectric spectroscopy at radiofrequencies (DS). In the framework of the Debye–Stokes–Einstein theory, the translational and rotational coefficients allow an estimation of hydrodynamic radius of the protein. A decoupling between translational and rotational dynamics, observed as a different estimation of hydrodynamic radius, is reported in the literature for some systems. In order to ascertain if this effect is present also in our sample, we performed PCS and DS measurements on lysozyme–water–glycerol solutions. The content of glycerol was in the range of 0–70% w/w, with a solvent viscosity from 0.9 to about 10 cpoise, and the protein concentration was up to 20 mg ml −1 . The average sizes of lysozyme, obtained by the two methods, are remarkably different at high protein concentrations. However, the values of hydrodynamic radius extrapolated to infinite dilution are coincident and independent of glycerol. These results indicate that the diffusive behavior of lysozyme in the water–glycerol mixture is coherent with the Debye–Stokes–Einstein hydrodynamic model.

Highly sensitive differential scanning microcalorimetry (DSC) has been used to investigate the phase transition properties of lipid vesicles prepared from 1,2-distearoyl-L-3-glyceryl-phosphatidylcholine (DSPC) in H(2)O and D(2)O. The data show that the response of pre-transition properties to D(2)O--H(2)O substitution is stronger than the main transition properties. We find that there is a small increase in the phase transition temperature (DeltaT approximately 0.5 K) and in the co-operative unit in the main transition. The increase in enthalpy (DeltaH congruent with1 kJ(.)mol(-1)) and in transition temperature (DeltaT congruent with2 K) observed in the pre-transition is comparable with that observed in quite different processes and systems, i.e. melting of nucleic acids and proteins and gel formation. It is suggested that D(2)O--H(2)O substitution affects the thermal transition in these systems in such a way that the contributions of enthalpy and entropy to structural reorganization of water in these processes is modified.

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differential scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the ‘classical’ twice NH4Cl-washed ribosomes, also known as loose couples (LC), or to the ‘tight couples’ preparative protocol (TC). Results show that 70S particles prepared according to the two different protocols exhibit different structural properties. Two subsequent relaxation processes occur in both samples as measured by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment. This is suggestive of a more extensive exposure of RNA to the solvent and of an overall more relaxed structure. The smaller LC subunit exhibits only one relaxation while the TC 30S shows two dielectric dispersions as well as 70S. No substantial differences were evidenced in either 50S species. Two typical melting peaks were observed by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles produce a large unstructured curve. On the basis of these results we conclude that TC 70S particles are more compact than LC ribosomes and that in the former ones the rRNA is less exposed to the solvent phase. Furthermore 30S particles obtained from TC show a more stable structure with respect to LC 30S. We conclude that the 30S subunit gives a major contribution to the compact character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosome species.

In this paper we show a microcalorimetric investigation carried out on the so-called cores, i. e. ribosomes deprived of select proteins by LiCl treatment. Thermal degradation of native ribosomes gives rise to two thermal transitions occurring at different temperatures. In the cores the high temperature peak persists even after treatment at very high ion strength (2 м LiCl). This strongly suggests the existence of a very stable structure that was previously observed also in particles treated with agents that hydrolyze the RNA moiety. The low temperature peak gradually but dramatically decreases even though it never disappears completely This indicates that the treatment to obtain ribosomal cores does not cause complete unfolding of the particle but only the destabilization of a structural three-dimensional domain present in native ribosomes. These data are discussed in the light of previous results obtained by dielectric spectroscopy and microcalorimetric studies on ribosomal particles.

Measurements of dielectric relaxation in water‐tert‐butanol mixtures have been performed by means of a frequency domain coaxial technique in the range 0.03–3 GHz, at 5 °C and for an alcohol mole fraction X ranging from 0 to 0.1. Experimental data give evidence for two relaxation processes whose relative amplitude and relaxation time depend on alcohol concentration. A two state model involving hydration and bulk water mutually exchanging at a finite rate is used to interpret dielectric data in the water rich region (X

The dielectric relaxation of water— n -butoxyethanol mixtures has been studied as a function of alcohol concentration in the water-rich region by means of a frequency domain coaxial technique in the range 10 MHz—3 GHz. Both the static dielectric constant and dielectric relaxation time exhibit an anomalous behaviour which further supports the existence of a butoxyethanol microphase above a critical concentration (mole fraction of cosolvent X *=0.026 at T =2°C). Experimental data have been analyzed in terms of a simple model in which water molecules are divided in two main groups, hydration and bulk water, mutually exchanging at a finite rate.

The adiabatic compressibility of aqueous solutions of alkali halides has been determined as a function of both concentration and temperature.A simple two-phase aqueous solution model based on the idea of overlapping water hydration shells is proposed to interpret the idea. The alkali halides examined exhibit a simple dependence of the size and the compressibility of the relevant shells or the ionic radii.

Different structural probes following the unfolding process of a protein may evidence the existence of stable intermediates from the differences in the unfolding curves. In this work, we analyze the thermal unfolding of chicken egg lysozyme in the presence of 8 M urea at pH 5 combining dielectric spectroscopy in the radiofrequency region, UV absorbance and circular dichroism in the far and near UV regions. The data are consistent with a two-state model of unfolding equilibrium and do not provide support for the presence of a significantly populated intermediate state.

The effect of ethanol on the critical micelle concentration (CMC) of several surfactant aqueous solutions has been studied by surface tension methods. The results show that with increasing alcohol concentration the CMC values first reach a minimum at an intermediate ethanol mole fraction x2* ≈ 0.055 and then increase with increasing x2. The value of x2* is close to that at which structural changes in the mixture occur as inferred from compressibility and optical absorption measurements. The present experiments support the assumption that the dominant mechanism by which ethanol affects the micellization process is through its effect on the structure of water.

— When DNA is complexed with Cu2+ ions it becomes sensitized to mid-UV light. Irradiation of the complex at 310 ± 10 nm produces marked changes in absorption and circular dichroism spectra in addition to those in melting profiles of irradiated samples. These variations show conformational changes in DNA structure due to a photoreduction of some copper sites in the DNA-Cu2+ complex. In addition to this effect an irreversible damage in DNA structure could be invoked to explain the observed behaviour. These facts can be related to the presence in DNA-Cu2+ complex of a small absorption band extending up to mid-UV range while the absorption of DNA is negligible in this spectral range.

The interaction of copper(II) with adenosine triphosphate (ATP) has been studied as a function of pH in the range pH 3–12. Our approach is the study of the effect of binding both on the ATP ultraviolet absorption spectrum and on the optical d-d transition of copper ions. The results show that Cu(II)-ATP complexes exist in a variety of forms in equilibrium, the percentage of each species varying according to the state of ionization of the intrinsic adenine, phosphate and ribose groups. These results also show a close correlation between the rate of dephosphorylation of ATP in the presence of CU(II) ions and Cu(II) bonding to the adenine of ATP, thus supporting the hypothesis that the metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion promoted dephosphorylation of ATP (D.H. Buisson and H. Sigel, Biochim. Biophys. Acta 343 (1974) 45).

A neutron-scattering investigation of the internal picosecond dynamics of lysozyme solvated in glycerol as a function of temperature in the range 200-410 K has been undertaken. The inelastic contribution to the measured intensity is characterized by the presence of a bump generally known as "boson peak", clearly distinguishable at low temperature. When the temperature is increased the quasielastic component of the spectrum becomes more and more intrusive and progressively overwhelms the vibrational bump. This happens especially for T345 K when the protein goes through an unfolding process, which leads to the complete denaturation. The quasielastic term is the superposition of two components whose intensities and linewidths have been studied as a function of temperature. The slower component describes motions with characteristic times of approximately 4 ps corresponding to reorientations of polypeptide side chains. Both the intensity and linewidth of this kind of relaxations show two distinct regimes with a crossover in the temperature range where the melting process occurs, thus suggesting the presence of a dynamical transition correlated to the protein unfolding. Conversely the faster component might be ascribed to the local dynamics of hydrogen atoms caged by the nearest neighbors with characteristic time of approximately 0.3 ps.

Through elastic neutron scattering we measured the mean-square displacements of the hydrogen atoms of lysozyme embedded in a glucose-water glassy matrix as a function of the temperature and at various water contents. The elastic intensity of all the samples has been interpreted in terms of the double-well model in the whole temperature range. The dry sample shows an onset of anharmonicity at approximately 100 K, which can be attributed to the activation of methyl group reorientations. Such a protein intrinsic dynamics is decoupled from the external environment on the whole investigated temperature range. In the hydrated samples an additional and larger anharmonic contribution is provided by the protein dynamical transition, which appears at a higher temperature Td. As hydration increases the coupling between the protein internal dynamics and the surrounding matrix relaxations becomes more effective. The behavior of Td that, as a function of the water content, diminishes by approximately 60 K, supports the picture of the protein dynamics as driven by solvent relaxations. A possible connection between the protein dynamical response versus T and the thermal stability in glucose-water bioprotectant matrices is proposed.

Metal ions are required for virtually all biological processes in which nucleic acids are engaged and play an essential role in their structural and dynamic properties. Nevertheless the presence of the “wrong” metal ions, or even the essential ones in the “wroog” concentration may cause harmful biological effects (toxic, mutagenic, carcinogenic).

The K-edge X-ray absorption near-edge structure (XANES) of a Cu(II) ion in NaOH solution (2 M) has been measured and compared with Cu(II) ions in aqueous solution. The spectra have been analyzed using multiple scattering theory. All spectral features have been interpreted in terms of one-electron transitions and the distance of axial ligands has been quantitatively determined byXANES. There is no need to invoke shake down or shake up effects at the Cu K-edge.

The structure of water in cetyltrimethylammonium bromide (CTABr) reverse micelles in dichloromethane has been studied as a function of W = [H2O]/[surfactant] by using the IR absorption due to O−H stretching modes in the 3800−3000 cm-1 range. We restricted our analysis to the region of small amounts of water (0 5. The rate effects can be ascribed to an increase in polarity and hydration of the reaction region as water is added and are related quantitatively to changes in the IR spectrum.