Contrasting dynamics of fragile and non-fragile polyalcohols through the glass, and dynamical, transitions: A comparison of neutron scattering and dielectric relaxation data for sorbitol and glycerol.

BACKGROUND: Glycerol and sorbitol are glass-forming hydrogen-bonded systems characterized by intriguing properties which make these systems very interesting also from the applications point of view. The goal of this work is to relate the hydrogen-bonded features, relaxation dynamics, glass transition properties and fragility of these systems, in particular to seek insight into their very different liquid fragilities. METHODS: The comparison between glycerol and sorbitol is carried out by collecting the elastic incoherent neutron scattering (EINS) intensity as a function of temperature and of the instrumental energy resolution. RESULTS: Intensity data vs temperature and resolution are analyzed in terms of thermal restraint and Resolution Elastic Neutron Scattering (RENS) approaches. CONCLUSIONS: The number of OH groups, which are related to the connecting sites, is a significant parameter both in the glass transition and in the dynamical transition. On the other hand, the disordered nature of sorbitol is confirmed by the existence of different relaxation processes. GENERAL SIGNIFICANCE: From the applications point of view, glycerol and sorbitol have remarkable bioprotectant properties which make these systems useful in different technological and industrial fields. Furthermore, polyols are rich in glassforming liquid phenomenology and highly deserving of study in their own right. The comparison of EINS and calorimetric data on glycerol and sorbitol helps provide a connection between structural relaxation, dynamical transition, glass transition, and fragility. The evaluation of the inflection point in the elastic intensity behavior as a function of temperature and instrumental energy resolution provides a confirmation of the validity of the RENS approach. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Protein dynamics as seen by (quasi) elastic neutron scattering.

BACKGROUND: Elastic and quasielastic neutron scattering studies proved to be efficient probes of the atomic mean square displacement (MSD), a fundamental parameter for the characterization of the motion of individual atoms in proteins and its evolution with temperature and compositional environment. SCOPE OF REVIEW: We present a technical overview of the different types of experimental situations and the information quasi-elastic neutron scattering approaches can make available. In particular, MSD can crucially depend on the time scale over which the averaging (building of the "mean") takes place, being defined by the instrumental resolution. Due to their high neutron scattering cross section, hydrogen atoms can be particularly sensitively observed with little interference by the other atoms in the sample. A few examples, including new data, are presented for illustration. MAJOR CONCLUSIONS: The incoherent character of neutron scattering on hydrogen atoms restricts the information obtained to the self-correlations in the motion of individual atoms, simplifying at the same time the data analysis. On the other hand, the (often overlooked) exploration of the averaging time dependent character of MSD is crucial for unambiguous interpretation and can provide a wealth of information on micro- and nanoscale atomic motion in proteins. GENERAL SIGNIFICANCE: By properly exploiting the broad range capabilities of (quasi)elastic neutron scattering techniques to deliver time dependent characterization of atomic displacements, they offer a sensitive, direct and simple to interpret approach to exploration of the functional activity of hydrogen atoms in proteins. Partial deuteration can add most valuable selectivity by groups of hydrogen atoms. "This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo".

Bio-protective effects of homologous disaccharides on biological macromolecules.

In this contribution the effects of the homologous disaccharides trehalose and sucrose on both water and hydrated lysozyme dynamics are considered by determining the mean square displacement (MSD) from elastic incoherent neutron scattering (EINS) experiments. The self-distribution function (SDF) procedure is applied to the data collected, by use of IN13 and IN10 spectrometers (Institute Laue Langevin, France), on trehalose and sucrose aqueous mixtures (at a concentration corresponding to 19 water molecules per disaccharide molecule), and on dry and hydrated (H(2)O and D(2)O) lysozyme also in the presence of the disaccharides. As a result, above the glass transition temperature of water, the MSD of the water-trehalose system is lower than that of the water-sucrose system. This result suggests that the hydrogen-bond network of the water-trehalose system is stronger than that of the water-sucrose system. Furthermore, by taking into account instrumental resolution effects it was found that the system relaxation time of the water-trehalose system is longer than that of the water-sucrose system, and the system relaxation time of the protein in a hydrated environment in the presence of disaccharides increases sensitively. These results explain the higher bioprotectant effectiveness of trehalose. Finally, the partial MSDs of sucrose/water and trehalose/water have been evaluated. It clearly emerges from the analysis that these are almost equivalent in the low-Q domain (0-1.7 Å(-1)) but differ substantially in the high-Q range (1.7-4 Å(-1)). These findings reveal that the lower structural sensitivity of trehalose to thermal changes is connected with the local spatial scale.

Study of solvent-protein coupling effects by neutron scattering.

The present work aims to characterize the dynamical behavior of proteins immersed in bio-preserving liquids and glasses. For this purpose, the protein dUTPase was chosen, while the selected solvents were glycerol, a triol, and some homologous disaccharides, i.e., trehalose, maltose, and sucrose, which are known to be very effective bio-preserving agents. The results highlight that the disaccharides show a slowing down effect on the water dynamics, which is stronger for trehalose than in the case of the other disaccharides. Furthermore, a characterization of the medium which hosts the protein is performed by using an operative definition of fragility based on the mean square displacement extracted by elastic incoherent neutron scattering, which is directly connected to Angell's kinetic fragility based on the viscosity. Finally, a study of the dynamics of the protein sequestered within the solvents is performed. The result shows that the protein dynamics is coupled with that of the surrounding matrix.

Resolution-elastic neutron scattering by correlation techniques.

Neutron scattering applications often require discriminating the elastic contribution from the inelastic contribution. For this purpose, correlation spectroscopy offers an effective tool with both pulsed and continuous neutron sources as well as several advantages: the analysis of the neutron velocity distribution can be carried out with a duty factor of 50%, independently on the resolution value; the best statistical accuracy for spectra where the elastic part encompasses most of the integrated intensity is provided. Depending on the statistical chopper position, correlation analysis can be used for both incoming and outgoing neutron velocity determination. Moreover, the correlation technique is very profitable for investigating weak signals in the presence of high background, which is often the case for small samples. To provide instrument flexibility and versatility, an innovative approach comprising tuning resolution by variable Resolution-Elastic Neutron Scattering (RENS) is proposed, offering further benefits by enabling systematic trading of intensity for resolution and vice versa. This study puts into evidence the advantages offered by the use of statistical chopper and of correlation technique for RENS in choosing the best compromise between resolution and beam intensity.

Study of the conformational behaviour of trehalose mycolates by FT-IR spectroscopy.

Mycolic acids are fundamental cell wall components, found in the outer membrane barrier (mycomembrane) of Mycobacterium related genera, that have shown antigenic, murine innate immunity inducting and inflammatory activity triggering action. The mycolic acid derivatives, such as the lipid extractable trehalose monomycolates (TMM) and dimycolates (TDM), have been extensively investigated by several biochemical and biological methods and, more recently, we have performed the first neutron scattering measurements on these molecules in order to characterize their dynamical behavior as well as their rigidity properties. In the present paper, we show the first systematic FT-IR study on TMM, TDM and glucose monomycolate (GMM). It includes the analysis of individual lipids but also mixtures of TMM/TDM (ratio of 1:1) or TMM/GMM (ratio of 1:2). The present work is aimed to the first characterization of the vibrational behavior of mycolates and their mixtures enabling us to elucidate the molecular mechanisms responsible for the capability of mycolic acids to affect the flexibility and permeability properties of the mycomembrane. As a whole, the present FT-IR findings provide information that have relevant biological implications, allowing to demonstrate that the membrane fluidity is not only linked to the chain length, but also to the specific conformational behavior adopted by mycolates, which in the mixtures is strongly affected by their mutual interactions. In addition, the capability of trehalose to drive the mycolate conformational behavior and then the chain order and packing is emphasized; due to the TDM relevant evidences shown by our data, this trehalose effect could be related to the TDM toxicity and inflammation action.

Changes in vibrational modes of water and bioprotectants in solution.

Inelastic neutron scattering (INS) measurements have been performed on trehalose and sucrose/H(2)O mixtures at very low temperature as a function of concentration by using the TOSCA spectrometer at the ISIS Facility (DRAL, UK). The aim of this work is to investigate by INS the vibrational behaviour of water in presence of trehalose and sucrose in order to characterize the changes induced by these disaccharides on the H(2)O hydrogen-bonded network. In particular, we obtained information about the effects of the two disaccharides in the translational, librational and bending spectral regions of ice. The disaccharide bioprotective effectiveness can be linked by the high destructuring effect emphasised by the analysis of the librational modes region. On the other hand, the analysis of the vibrational region corresponding to the ice bending modes show a high "crystallinity" degree which can justify the cryptobiotic action of disaccharides.

Concentration dependence of vibrational properties of bioprotectant/water mixtures by inelastic neutron scattering.

Neutron scattering has been demonstrated to be a powerful tool for characterizing the structure and dynamics of biological molecules and for investigating the physical and chemical mechanisms of biophysical processes. The aim of the present work is to investigate by inelastic neutron scattering (INS) the vibrational behaviour of a class of bioprotectant systems, such as homologous disaccharides, trehalose, maltose and sucrose, in water mixtures. INS measurements have been performed on trehalose/H2O, maltose/H2O and sucrose/H2O mixtures at very low temperature as a function of concentration by using the thermal original spectrometer with cylindrical analyzers (TOSCA) spectrometer at the ISIS Facility (DRAL, UK). The findings allow the analyses of the vibrational features of the INS spectra in order to study the effect of disaccharides on the H2O hydrogen-bonded tetrahedral network. The obtained neutron scattering findings point out that disaccharides, and in particular trehalose, have a destructuring effect on the water tetrahedral network, as emphasized by the analysis of the librational modes region from 50 to 130 meV energy transfer. On the other hand, the analysis of the bending modes region (130-225 meV) shows a locally ordered structure in the disaccharide/H2O mixtures.Finally, the observed experimental evidences are linked to the different bioprotective effectiveness of disaccharides as a function of concentration.

Macro- and microdefinitions of fragility of hydrogen-bonded glass-forming liquids.

In the present paper, the nature of the fragility of liquids with a developed H-bond network is investigated. It is shown that the effective activation energy of the shear viscosity eta and the fragility parameter defined as m=d log eta/d(Tg/T)|t=t+g are immediately connected with the average number nH(T) of the H-bond per molecule. The consistency of the macroscopic (m) and the microscopic [M=d(u(2)(0)/2)>loc)/d(Tg/T)|T=T+8, where 2>loc=2>anharm - 2>harm] definitions of the fragility is established.

How homogeneous are the trehalose, maltose, and sucrose water solutions? An insight from molecular dynamics simulations.

The structural properties resulting from the reciprocal influence between water and three well-known homologous disaccharides, namely, trehalose, maltose, and sucrose, in aqueous solutions have been investigated in the 4-66 wt % concentration range by means of molecular dynamics computer simulations. Hydration numbers clearly show that trehalose binds to a larger number of water molecules than do maltose or sucrose, thus affecting the water structure to a deeper extent. Two-dimensional radial distribution functions of trehalose solutions definitely reveal that water is preferentially localized at the hydration sites found in the trehalose dihydrate crystal, this tendency being enhanced when increasing trehalose concentration. Over a rather wide concentration range (4-49 wt %), the fluctuations of the radius of gyration and of the glycosidic dihedral angles of trehalose indicate a higher flexibility with respect to maltose and sucrose. At sugar concentrations between 33 and 66 wt %, the mean sugar cluster size and the number of sugar-sugar hydrogen bonds formed within sugar clusters reveal that trehalose is able to form larger clusters than sucrose but smaller than maltose. These features suggest that trehalose-water mixtures would be more homogeneous than the two others, thus reducing both desiccation stresses and ice formation.

Inelastic neutron scattering study on bioprotectant systems.

We collected inelastic neutron scattering (INS) spectra of homologous disaccharide (C12H22O11)/H2O mixtures at a very low temperature by using indirect geometry time-of-flight spectrometer TOSCA at the ISIS pulse neutron facility (DRAL, UK). The aim of this work is to investigate the vibrational behaviour of trehalose, maltose and sucrose/H2O mixtures with INS in order to characterize the structural changes induced by these disaccharides on the H2O hydrogen-bonded network. A higher degree of 'crystallinity' for the trehalose/H2O system is observed in the vibrational region corresponding to the ice bending modes. This feature could justify the better cryptobiotic action of trehalose compared with maltose and sucrose. On the other hand, the better bioprotective effectiveness could be explained by the higher destructuring effect of trehalose, emphasized by the analysis of the librational modes region.

Correlation between bioprotective effectiveness and dynamic properties of trehalose-water, maltose-water and sucrose-water mixtures.

The aim of the present work is to link the bioprotective effectiveness to the dynamic properties of a class of homologous disaccharides, that is, trehalose, maltose and sucrose, and their mixtures in water. The findings obtained by elastic neutron scattering point out a harmonic-anharmonic transition for all the three disaccharide mixtures. Using a new operative definition of 'fragility', the different degrees of 'strength' of the investigated systems are determined. The links existing between the degree of fragility and the cryptoprotective action are also discussed.

FTIR, ESI-MS, VT-NMR and SANS study of trehalose thermal stabilization of lysozyme.

Protein aggregation is often associated with conformational and structural changes of secondary structure elements that may lead to exposure of some specific residues. Data obtained in our experimental work indicate that trehalose (1.0M) effectively prevent thermal inactivation and aggregation of lysozyme. In fact, following heat treatment, lysozyme generates insoluble aggregates which are almost completely absent in the samples incubated in the presence of the disaccharide. The experimental approach consists in studying FTIR spectra of intrinsic chromophores and VT-NMR measurements on lysozyme water mixtures in the presence of trehalose. FTIR measurements suggest that in the presence of 1.0 M of trehalose there is a clear decrease in the loss of α-helix structure and in the formation of intermolecularly aggregated structures. Electrospray ionization mass spectrometry (ESI-MS) was employed to characterize protein structural transition, highlighting as trehalose remarkably influenced solvent accessibility to the amide peptide backbone upon heat treatment, consequentially decreasing local protein environment changes. Complementary informations are also obtained by UV-vis spectroscopy measurements, Congo Red binding and activity determinations.

Innovative wavelet protocols in analyzing elastic incoherent neutron scattering.

Wavelet analysis has recently found a wide range of applications in Physics, Mathematics, and signal processing. This is mainly due to its ability to locally resolve a nonstationary signal in terms of functional forms, called mother wavelets, and to firmly locate trend anomalies in the signal. In the present paper, some examples of the application of wavelet analysis to elastic incoherent neutron scattering (EINS) data collected by the IN13 spectrometer at the Institute Laue Langevin (ILL) on water mixtures of the three homologous disaccharides, trehalose, maltose, and sucrose, and on literature data of dry and hydrated lysozyme and myoglobine as a function of temperature and of exchanged wave vector are presented. The experimental findings have been analyzed by means of a wavelet analysis that allows one to characterize the scattered elastic intensity behavior on different scales and to locate the discontinuities and the trend anomalies in the registered signal. This latter procedure is made possible thanks to the multiscale analysis, which allows, by decreasing the scale, one to localize the peculiar trend features. The entire body of the experimental findings reveals different transition temperatures for the three investigated disaccharides together with a stronger temperature dependence of the maltose/H(2)O and sucrose/H(2)O systems in comparison with the trehalose/H(2)O mixture, which signals a stronger character of this latter in comparison with the other two homologous disaccharides. These results justify the better ability of trehalose, with respect to maltose and sucrose, to encapsulate biostructures in a more rigid matrix.

Stabilization effects of kosmotrope systems on ornithine carbamoyltransferase.

In the present article the influence of salts and additives, such as trehalose, NaCl, ornithine, sodium phosphate and ammonium sulphate, on ornithine carbamoyltransferase (OCTase) is investigated in order to study the OCTase stabilization process as a function of solutes and to point out the fundamental role played by an enhancement of hydrophobic interactions. The synergic use of different techniques, such as neutron spectroscopy, UV-vis spectroscopy, activity and thermal measurements, allows to highlight the cosolute capability to avoid thermal inactivation, to induce important changes in secondary and tertiary enzyme structure and to stabilize biological macromolecules.

Study of the dynamical properties of water in disaccharide solutions.

This work presents quasi-elastic neutron scattering (QENS) and neutron spin echo (NSE) findings on homologous disaccharide (i.e. trehalose, maltose and sucrose)/water solutions as a function of temperature. The dynamical properties of these systems are investigated by QENS, which, on the picosecond scale, allows for the characterisation of the diffusion of both solutes and solvent. On the other hand, NSE investigates the dynamics on the nanosecond scale, allowing for the relaxation times of the disaccharide/water systems to be evaluated. The experimental data highlight a strong slowing down of water in the presence of disaccharides. The whole set of findings indicates, therefore, a noticeable disaccharide-water interaction, which is more intense in the case of trehalose. This feature can justify its higher bioprotective effectiveness.

Fragility by elastic incoherent neutron scattering.

The present work furnishes an operative definition for the fragility degree by using elastic incoherent neutron scattering (EINS). Such a definition is based on the relation between viscosity, a macroscopic quantity, and the atomic mean-square displacement, which refers to a nanoscopic property. This procedure has been used to analyze a set of glass-forming systems and it allows to obtain a linear dependence of the fragility parameter M, obtained by EINS, on the fragility parameter m, obtained by viscosity measurements.

Mean-square displacement relationship in bioprotectant systems by elastic neutron scattering.

Neutron intensity elastic scans on trehalose, maltose, and sucrose/H(2)O mixtures as a function of concentration, temperature, and exchanged wave vector are presented. The experimental findings show a crossover in molecular fluctuations between harmonic and anharmonic dynamical regimes. A new operative definition for the degree of fragility of glass-forming systems is furnished by using explicitly the connection between viscosity and mean-square displacement. The procedure is tested for the investigated mixtures and for a set of glass-forming systems. In this frame, the stronger character of trehalose/H(2)O mixture indicates a better attitude in respect to maltose and sucrose/H(2)O mixtures to encapsulate biostructures in a more rigid matrix.