Assunta Morresi

Hydrogen bond dynamics and water structure in glucose-water solutions by depolarized Rayleigh scattering and low-frequency Raman spectroscopy

The effect of glucose on the relaxation process of water at picosecond time scales has been investigated by depolarized Rayleigh scattering (DRS) experiments. The process is assigned to the fast hydrogen bonding dynamics of the water network. In DRS spectra this contribution can be safely separated from the slower relaxation process due to the sugar. The detected relaxation time is studied at different glucose concentrations and modeled considering bulk and hydrating water contributions. As a result, it is found that in diluted conditions the hydrogen bond lifetime of proximal water molecules becomes about three times slower than that of the bulk. The effect of the sugar on the hydrogen bond water structure is investigated by analyzing the low-frequency Raman (LFR) spectrum sensitive to intermolecular modes. The addition of glucose strongly reduces the intensity of the band at 170 cm(-1) assigned to a collective stretching mode of water molecules arranged in cooperative tetrahedral domains. These findings indicate that proximal water molecules partially lose the tetrahedral ordering typical of the bulk leading to the formation of high density environments around the sugar. Thus the glucose imposes a new local order among water molecules localized in its hydration shell in which the hydrogen bond breaking dynamics is sensitively retarded. This work provides new experimental evidences that support recent molecular dynamics simulation and thermodynamics results.

Broadband Depolarized Light Scattering Study of Diluted Protein Aqueous Solutions

A broadband depolarized light scattering (DLS) study is performed on diluted lysozyme aqueous solutions as a function of temperature and concentration. The dynamical susceptibility, obtained in a wide spectral range (0.6-36000 GHz) through the coupled use of interferometric and dispersive devices, is interpreted and compared with neutron scattering and Raman-induced optical Kerr-effect literature data, thus giving a general picture of relaxation phenomena. We show that the proposed approach represents a suitable tool for investigating the hydration dynamics of protein-water solutions. A detailed analysis of the quasi-elastic scattering region evidences the existence of two distinct relaxational processes at picosecond time scales. The fast process (fractions of picosecond) is attributed to bulk water dynamics, while the slow one (few picoseconds) is attributed to dynamical rearrangements of water molecules strongly influenced by the protein (hydration water). The retardation effect here estimated of about 6-7 can be regarded as a direct measure of the increased protein-water and water-water hydrogen bond stability of the water molecules within the protein hydration shell. Interestingly, a similar effect was previously observed on small hydrophilic sugar molecules. Moreover, backbone and side chains torsional motions of the protein in the 600-5300 GHz frequency range are found to be insensitive to thermal variations and to eventual changes occurring in the premelting zone.

More Is Different: Experimental Results on the Effect of Biomolecules on the Dynamics of Hydration Water.

Biological interfaces characterized by a complex mixture of hydrophobic, hydrophilic, or charged moieties interfere with the cooperative rearrangement of the hydrogen-bond network of water. In the present study, this solute-induced dynamical perturbation is investigated by extended frequency range depolarized light scattering experiments on an aqueous solution of a variety of systems of different nature and complexity such as small hydrophobic and hydrophilic molecules, amino acids, dipeptides, and proteins. Our results suggest that a reductionist approach is not adequate to describe the rearrangement of hydration water because a significant increase of the dynamical retardation and extension of the perturbation occurs when increasing the chemical complexity of the solute.

Extended frequency range depolarized light scattering study of N-acetyl-leucine-methylamide-water solutions.

We have studied the influence of the amphiphilic model peptide N-acetyl-leucine-methylamide (NALMA) on the dynamics of water using extended frequency range depolarized light scattering (EDLS), between 0.3 GHz and 36 THz. This technique allowed us to separate solute from solvent dynamics and bulk from hydration water, providing both characteristic times and relative fractions. In the temperature range 5-65 °C, a retardation factor from 9 to 7 is found for water hydrating NALMA. Moreover, in the same range, a hydration number from 62 to 50 is observed, corresponding to more than two hydration layers. This strong perturbation suggests the existence of a collective effect of amphiphilic molecules on surrounding water molecules.

Spectroscopic study of acrylic resins in solid matrices

Abstract The degradation of the acrylic polymers, usually used as protective coatings in porous materials of artistic interest, determines depolymerization and/or cross-linking processes dangerous for the ‘protected’ materials. Three types of largely used commercially products have been applied as thin films on different specimens; their aging, induced with an exposition to a Xenon lamp, has been monitored with UV-VIS fluorimetry, microRaman and ATR (Attenuated Total Reflectance) techniques. The fluorimetric properties of the acrylic polymers make possible the application of the reflectance fluorescence for the control of cleaning procedures and for the monitoring of the conservation of the art objects. The microRaman technique is here used for the first time for the characterization of these coatings, giving the possibility to examine surfaces of order of magnitudes of microns. The comparison of the microRaman results with the ATR ones, gives a good, initial picture of the existing degradation processes, allowing to follow the evolution of some chosen vibrational bands, directly related to the changing of the molecular structure.

Unfolding and aggregation of lysozyme: a thermodynamic and kinetic study by FTIR spectroscopy.

The unfolding of hen egg-white lysozyme dissolved both in D(2)O and CH(3)CH(2)OD/D(2)O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event.

Vibrational circular dichroism spectra of lysozyme solutions: solvent effects on thermal denaturation processes.

Vibrational spectroscopy has been applied to the study of conformational variation of lysozyme during thermal denaturation. An infrared and vibrational circular dichroism (VCD) analysis of lysozyme in D2O, D2O/EtOD, and D2O/DMSO at different solvent compositions and pHs was accomplished. Complete deuteration effects on amidic groups were revealed through the analysis of the amide I band of lysozyme dissolved in deuterated water. The comparison of IR absorption and VCD spectra of lysozyme in D2O revealed the higher sensitivity of the VCD technique to the presence of partially or fully deuterated secondary structure of lysozyme. Aggregation was induced by thermalization of lysozyme at different solvent compositions and pH values. Interestingly, IR and VCD showed different sensitivities to reveal intermolecular β-sheet aggregation under thermal denaturation. The VCD intensity enhancement was observed as the aggregate dimension increased, whereas IR was sensitive to the nucleation step of the process. Moreover, chirality of supramolecular species was revealed through the analysis of the VCD sign pattern. For the first time, we demonstrate how aggregates produced under different solvent compositions but similar pHs showed the same VCD sign pattern and consequently the same sense of growth.

Molecular dynamics and vibrational relaxations in liquid nitromethane. II. Raman, coherent anti‐Stokes Raman spectroscopy, and transient optical Kerr effects in the totally symmetric ν4 mode in CH3NO2

The vibrational relaxation of the ν4 mode of CH3NO2 liquid and its molecular dynamics are investigated. Temperature and solvent effects on vibrational dephasing and correlation times are discussed in terms of the adiabatic impact theory to infer that, in the molecular collision, the attractive part of intermolecular potential is explored. The statistical uncorrelation of rotational and vibrational relaxations is experimentally demonstrated.

Raman micro-spectroscopy: A powerful tool for the monitoring of dynamic supramolecular changes in living cells

Cellular imaging techniques have become powerful tools in cell biology. With respect to others, the techniques based on vibrational spectroscopy present a clear advantage: the molecular composition and the modification of subcellular compartments can be obtained in label-free conditions. In fact, from the evolution of positions, intensities and line widths of Raman and infrared bands in the cell spectra, characteristic information on cellular activities can be achieved, and particularly, cellular death can be investigated. In this work we present the time evolution of the Raman spectra of single live Jurkat cells (T-lymphocyte) by looking at the high frequency part of their Raman spectra, that is the CH stretching region, around 3000cm(-1). In particular, investigation into the composition or rearrangement of CH bounds, markers of cellular membrane fatty acids, can represent an important method to study and to recognize cell death. The experimental procedure we used, together with the analysis of these high frequency vibrational bands, may represent a new, improved and advantageous approach to this kind of study.

Denaturation and Preservation of Globular Proteins: The Role of DMSO

The thermal denaturation of hen egg white lysozyme (HEWL) in D(2)O was followed by IR absorption after addition of dimethyl sulfoxide (DMSO) at different molar fractions. Amide I intensity and position revealed that DMSO reduces the thermal stability of the native protein and favors the formation of ordered aggregates. The comparison with ethanol/water solutions evidenced that ethanol (partially deuterated ethanol EtOD) has a stronger effect on the thermal stability of HEWL: the same down-shift of melting temperature was measured at 0.18 and 0.30 molar fraction of ethanol and DMSO, respectively. This is probably due to lower polarity of EtOD/D(2)O with respect to DMSO/D(2)O solutions. A kinetic study of protein assembling at 0.30 DMSO molar fraction, was also performed at different temperatures. The high viscosity of the solvent was observed to cause a sensitive slowing down of aggregation rate in comparison to that of water/alcohol solutions. The evidence of a retarded self-assembling put forward a possible explanation for the use of dimethyl sulfoxide as a protectant of protein structure. In fact, for both organic solvents a nonspecific interaction with the protein and a water-mediated action is deduced, but the addition of DMSO reduces the irreversible denaturation due to kinetic effects and this can be exploited for lessening one of the main degradation routes of globular proteins during freezing-thawing cycles.