Stefania Perticaroli

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.

Vibrational Density of States of Hydration Water at Biomolecular Sites: Hydrophobicity Promotes Low Density Amorphous Ice Behavior

Inelastic neutron scattering experiments and molecular dynamics simulations have been used to investigate the low frequency modes, in the region between 0 and 100 meV, of hydration water in selected hydrophilic and hydrophobic biomolecules. The results show changes in the plasticity of the hydrogen-bond network of hydration water molecules depending on the biomolecular site. At 200 K, the measured low frequency density of states of hydration water molecules of hydrophilic peptides is remarkably similar to that of high density amorphous ice, whereas, for hydrophobic biomolecules, it is comparable to that of low density amorphous ice behavior. In both hydrophilic and hydrophobic biomolecules, the high frequency modes show a blue shift of the libration mode as compared to the room temperature data. These results can be related to the density of water molecules around the biological interface, suggesting that the apparent local density of water is larger in a hydrophilic environment.

Hydration and Aggregation in Mono- and Disaccharide Aqueous Solutions by Gigahertz-to-Terahertz Light Scattering and Molecular Dynamics Simulations

The relaxation properties of hydration water around fructose, glucose, sucrose, and trehalose molecules have been studied by means of extended frequency range depolarized light scattering and molecular dynamics simulations. Evidence is given of hydration dynamics retarded by a factor ξ = 5-6 for all the analyzed solutes. A dynamical hydration shell is defined based on the solute-induced slowing down of water mobility at picosecond time scales. The number of dynamically perturbed water molecules N(h) and its concentration dependence have been determined in glucose and trehalose aqueous solutions up to high solute weight fractions (ca. 45%). For highly dilute solutions, about 3.3 water molecules per sugar hydroxyl group are found to be part of the hydration shell of mono- and disaccharide. For increasing concentrations, a noticeable solute-dependent reduction of hydration number occurs, which has been attributed, in addition to simple statistical shells overlapping, to aggregation of solute molecules. A scaling law based on the number of hydroxyl groups collapses the N(h) concentration dependence of glucose and trehalose into a single master plot, suggesting hydration and aggregation properties independent of the size of the sugar. As a whole, the present results point to the concentration of hydroxyl groups as the parameter guiding both sugar-water and sugar-sugar interactions, without appreciable difference between mono- and disaccharides.

Mechanical Properties of Nanoscopic Lipid Domains

The lipid raft hypothesis presents insights into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. As a result, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approaches with inelastic neutron scattering, we isolate the bending modulus of ∼13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. From additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.

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.

Dynamics of hydration water in sugars and peptides solutions.

We analyzed solute and solvent dynamics of sugars and peptides aqueous solutions using extended depolarized light scattering (EDLS) and broadband dielectric spectroscopies (BDS). Spectra measured with both techniques reveal the same mechanism of rotational diffusion of peptides molecules. In the case of sugars, this solute reorientational relaxation can be isolated by EDLS measurements, whereas its contribution to the dielectric spectra is almost negligible. In the presented analysis, we characterize the hydration water in terms of hydration number and retardation ratio ξ between relaxation times of hydration and bulk water. Both techniques provide similar estimates of ξ. The retardation imposed on the hydration water by sugars is ~3.3 ± 1.3 and involves only water molecules hydrogen-bonded (HB) to solutes (~3 water molecules per sugar OH-group). In contrast, polar peptides cause longer range perturbations beyond the first hydration shell, and ξ between 2.8 and 8, increasing with the number of chemical groups engaged in HB formation. We demonstrate that chemical heterogeneity and specific HB interactions play a crucial role in hydration dynamics around polar solutes. The obtained results help to disentangle the role of excluded volume and enthalpic contributions in dynamics of hydration water at the interface with biological molecules.

Rotational dynamics of trehalose in aqueous solutions studied by depolarized light scattering

High resolution depolarized light scattering spectra, extended from 0.5 to 2x10(4) GHz by the combined used of a dispersive and an interferometric setup, give evidence of separated solute and solvent dynamics in diluted trehalose aqueous solutions. The slow relaxation process, located in the gigahertz frequency region, is analyzed as a function of temperature and concentration and assigned to the rotational diffusion of the sugar molecule. The results are discussed in comparison with the data obtained on glucose solutions and they are used to clarify the molecular origin of some among the several relaxation processes reported in literature for oligosaccharides solutions. The concentration dependence of relaxation time and of shear viscosity are also discussed, suggesting that the main effect of carbohydrate molecules on the structural relaxation of diluted aqueous solutions is the perturbation induced on the dynamics of the first hydration shell of each solute molecule.

Rigidity, secondary structure, and the universality of the boson peak in proteins.

Complementary neutron- and light-scattering results on nine proteins and amino acids reveal the role of rigidity and secondary structure in determining the time- and lengthscales of low-frequency collective vibrational dynamics in proteins. These dynamics manifest in a spectral feature, known as the boson peak (BP), which is common to all disordered materials. We demonstrate that BP position scales systematically with structural motifs, reflecting local rigidity: disordered proteins appear softer than α-helical proteins; which are softer than β-sheet proteins. Our analysis also reveals a universal spectral shape of the BP in proteins and amino acid mixtures; superimposable on the shape observed in typical glasses. Uniformity in the underlying physical mechanism, independent of the specific chemical composition, connects the BP vibrations to nanometer-scale heterogeneities, providing an experimental benchmark for coarse-grained simulations, structure/rigidity relationships, and engineering of proteins for novel applications.

Secondary structure and rigidity in model proteins

There is tremendous interest in understanding the role that secondary structure plays in the rigidity and dynamics of proteins. In this work we analyze nanomechanical properties of proteins chosen to represent different secondary structures: α-helices (myoglobin and bovine serum albumin), β-barrels (green fluorescent protein), and α + β + loop structures (lysozyme). Our experimental results show that in these model proteins, the β motif is a stiffer structural unit than the α-helix in both dry and hydrated states. This difference appears not only in the rigidity of the protein, but also in the amplitude of fast picosecond fluctuations. Moreover, we show that for these examples the secondary structure correlates with the temperature- and hydration-induced changes in the protein dynamics and rigidity. Analysis also suggests a connection between the length of the secondary structure (α-helices) and the low-frequency vibrational mode, the so-called boson peak. The presented results suggest an intimate connection of dynamics and rigidity with the protein secondary structure.

Dynamics and Rigidity in an Intrinsically Disordered Protein, β‑Casein

The emergence of intrinsically disordered proteins (IDPs) as a recognized structural class has forced the community to confront a new paradigm of structure, dynamics, and mechanical properties for proteins. We present novel data on the similarities and differences in the dynamics and nanomechanical properties of IDPs and other biomacromolecules on the picosecond time scale. An IDP, β-casein (CAS), has been studied in a calcium bound and unbound state using neutron and light scattering techniques. We show that CAS partially folds and stiffens upon calcium binding, but in the unfolded state, it is softer than folded proteins such as green fluorescence protein (GFP). We also see that some localized diffusive motions in CAS have a larger amplitude than in GFP at this time scale but are still smaller than those observed in tRNA. In spite of these differences, CAS dynamics are consistent with the classes of motions seen in folded protein on this time scale.