Daniela Russo

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.

The impact of hydration water on the dynamics of side chains of hydrophobic peptides: From dry powder to highly concentrated solutions

Elastic and quasielastic neutron scattering experiments are used to investigate the dynamics of side chains in proteins, using hydrophobic peptides, from dry and hydrated powders up to solutions, as models. The changes of the internal dynamics of a prototypical hydrophobic amino acid, N-acetyl-leucine-methylamide, and alanine amino acids are investigated as a function of water/peptide molecular ratio. While previous results have shown that, in concentrated solution, when the hydrophobic side chains are hydrated by a single hydration water layer, the only allowed motions are confined and can be attributed to librational/rotational movements associated with the methyl groups. In the present work we observe a dynamical evolution from dry to highly hydrated powder. We also observe rotational and diffusive motions and a dynamical transition at approximately 250 K for long side chain peptides while for peptides with short side chains, there is no dynamical transition but only rotational motions. With a local measurement of the influence of hydration water dynamics on the amino acid side chains dynamics, we provide unique experimental evidence that the structural and dynamical properties of interfacial water strongly influence the side chain dynamics and the activation of diffusive motions. We also emphasize that the side chain length has a role on the onset of dynamical transition.

Hydration water dynamics of a completely hydrophobic oligopeptide

Abstract The dynamics of hydration water of a completely deuterated penta-alanine peptide has been studied by incoherent quasi-elastic neutron scattering. Measurements have been made at different hydration levels (7%, 30%, 50%, 90%), and on the dried powder (0%) which contains one structural water molecule. The dynamical contribution of this first hydration molecule of water is characteristic of a slow rotational motion with a relaxation time, τ1, of 2.2 ps, similar to what is found in supercooled water dynamics. Adding two more hydration water molecules (7%) the rotational motion of the first water is coupled with the new diffusive motion and the dynamics profile can be, in first approximation, described through a rotational jump model. The results suggest a behavior similar to that of bulk water at 2 °C. At higher levels of hydration, the mobility of new molecules of water approaches that of bulk water, with a rotation relaxation time of 1 ps and a confined diffusing motion. However the residence time value, τ0, is of the same order of magnitude as supercooled water at T=−10 ° C.

Evidence of dynamical constraints imposed by water organization around a bio-hydrophobic interface.

Molecular dynamics simulations and elastic neutron scattering experiments have been used to highlight how the structural organization of hydration water is able in some cases to locally constrain atomic movements at biologic interfaces. Using fully hydrated small peptides as models of protein interfaces, we show that the length of the side chains and the hydrophilic backbone have specific signatures. The dynamics of the side chain, which is part of biomolecules, have not only a crucial role in the whole flexibility as compared to the backbone, but also modify the values of transition temperatures. The analysis of the activation energies of methyl group dynamics suggests that the interaction between hydrophobic side chain and surrounding water plays an important role in the whole flexibility as well. We suggest that the progressive water cluster organization, around hydrophobic interfaces increases the activation energy and that a plateau regime is reached only when an extended hydrogen-bond network is established. The cluster size corresponds to a single layer of water molecules.

Investigation into the Relaxation Dynamics of Polymer-Protein Conjugates Reveals Surprising Role of Polymer Solvation on Inherent Protein Flexibility.

Fully biodegradable protein-polymer conjugates, namely, MBP-PMeEP (maltose binding protein-poly methyl-ethylene phosphonate), have been investigated in order to understand the role of polymer solvation on protein flexibility. Using elastic and quasi-elastic incoherent neutron scattering, in combination with partially deuterated conjugate systems, we are able to disentangle the polymer dynamics from the protein dynamics and meaningfully address the coupling between both components. We highlight that, in the dry state, the protein-polymer conjugates lack any dynamical transition in accordance with the generally observed behavior for dry proteins. In addition, we observe a larger flexibility of the conjugated protein, compared to the native protein, as well as a lack of polymer-glass transition. Only upon water hydration does the conjugate recover its dynamical transition, leading to the conclusion that exclusive polymer solvation is insufficient to unfreeze fluctuations on the picosecond-nanosecond time scale in biomolecules. Our results also confirm the established coupling between polymer and protein dynamics in the conjugate.

Brillouin Neutron Spectroscopy as a Probe to Investigate Collective Density Fluctuations in Biomolecules Hydration Water

The role of water in the behaviour of biomolecules is well recognized. The coupling of motions between water and biomolecules has been studied in a wide time scale for the self part while collective dynamics is still quite unexplored. Self-dynamics provides information about the diffusion processes of water molecules and relaxation processes of the protein structure. Collective density fluctuations might provide important insight on the transmission of information possibly correlated to biological functionality. The idea that hydration water layers surrounding a biological molecule show a self-dynamical signature that differs appreciably from that of bulk water, in analogy with glass-former systems, is quite accepted. In the same picture Brillouin terahertz spectroscopy has been used to directly probe collective dynamics of hydration water molecules around biosystems, showing a weaker coupling and a more bulklike behaviour. We will discuss results of collective modes of hydration water, arising from neutron Brillouin spectroscopy, in the context of biomolecules-solvent interaction.

Reversible Bioconjugation: Biodegradable Poly(phosphate)-Protein Conjugates

Protein-polymer conjugates are widely used to improve the pharmacokinetic properties of therapeutic proteins. Commercially available conjugates employ poly(ethylene glycol) (PEG) as the protective polymer; however, PEG has a number of shortcomings, including non-biodegradability and immunogenicity, that call for the development of alternatives. Here, the synthesis of biodegradable poly(phosphate), that is, poly(ethyl ethylene phosphate) (PEEP), by organo-catalyzed anionic ring-opening polymerization exhibiting dispersity values Ð < 1.3 is reported. Polymers with molecular weights between 2000 and 33 200 g mol-1 are then ω-functionalized with a succinimidyl carbonate group and subsequently conjugated to model proteins. These are the first conjugates based on polyphosphates which degraded upon exposure to phosphodiesterase. As is the case for PEGylated therapeutics, residual in vitro activity of the PPEylated conjugates depends on the extent of protein modification. These results suggest that PEEP exhibits the desired properties of a biopolymer for use in next generation, fully degradable drug delivery systems.

Water Collective Dynamics in Whole Photosynthetic Green Algae as Affected by Protein Single Mutation

In the context of the importance of water molecules for protein function/dynamics relationship, the role of water collective dynamics in Chlamydomonas green algae carrying both native and mutated photosynthetic proteins has been investigated by neutron Brillouin scattering spectroscopy. Results show that single point genetic mutation may notably affect collective density fluctuations in hydrating water providing important insight on the transmission of information possibly correlated to biological functionality. In particular, we highlight that the damping factor of the excitations is larger in the native compared to the mutant algae as a signature of a different plasticity and structure of the hydrogen bond network.