Slow dynamics in supercooled aqueous solutions for cryopreservation : a molecular dynamics study

Abstract

The aim of this thesis is to characterize some of the properties of supercooled water in order to better understand its behaviour in the supercooled region and how to exploit these properties for cryopreservation of biomolecules [1, 2]. Water is one of the most common element on Earth and plays a key role in many different and important physical, chemical and biological processes. A deep understanding of the mechanisms that take place in supercooled water may lead to many progresses in all these fields. This work aimed at the description of supercooled water in biological solutions, with focus on the population of water molecules that resides in the hydration layer of the biomolecule under investigation. Using highly optimized software to perform MD simulations and analysis of the resulting trajectories, five systems have been studied from a dynamical and structural point of view. Two of them are aqueous solutions of trehalose with different concentration of the disaccharide. The other three are composed by water, lysozyme DMSO and trehalose in different combinations. Particular at tention has been posed on the characterization of the water-water hydrogen bonds network and on the dynamics of hydration water in the Mode Coupling Theory framework [3]. Trehalose has been found to be very effective in modify the dynamics of water molecules at its interface. The structural α relaxation typical of glassy system is present also in water population in the hydration layer, with a slight modification of the phenomenology seen in bulk water. In particular the Fragile to-Strong Crossover is preserved, with a shift of the temperature of transition to an higher value. This suggests that aqueous solutions can be a feasible way to enter the no man’s land and study anomalies of water in a region where experiments on bulk water are difficult. In addition to the α relaxation, a slow relaxation has been observed in the long time region of the Self Intermediate Scattering Functions. This new feature is connected with the interaction of water molecules with trehalose molecules and it is not present in bulk water. The study of this new relaxation process leads to a connection with fluctua tions of the clusters formed by trehaloses and a complete dynamical coupling between aggregates of trehalose molecules and water molecules at the interface is observed. A dynamical transition is found at around 250-260 K, akin to what is observed in hydrated protein upon heating. This is the first evidence of a dynamical transition in a self-assembled macroaggregate composed of small biomolecules. Trehalose molecules have also a strong impact on the formation of water-water HBs. In particular a change in the number of HBs per water molecule has been observed. Moreover the nearby presence of trehalose molecules change also the spatial arrangement of water molecules, with a modification of the typical tetra hedral structure of the HBs network observed in bulk water. These evidences are all connected to trehalose behaving as a good cryoprotec tant, it is able to hinder the motion of surrounding water molecules and to hamper the formation of ice crystal upon cooling. Analysis of the MD simulations concerning the composite mixtures of DMSO and trehalose show effects on both the dynamics and structure of water molecules in the hydration shell of lysozyme protein. The phenomenology typical of glassy water is retained also in these multicompo nent systems. The characteristic long relaxation time shows a strong to strong crossover upon changing the temperature. This feature has been observed in all the biological solutions under investigation. The crossover temperature de pends on the composition of the mixture: it is higher in the two solutions with trehalose. To enquire on the hypothesis of a coupling between the large time motion of a protein and that of the surrounding water, the MSDs of the oxygen atoms of hy dration water and the RMSD of hydrogen atoms of the side chains of lysozyme have been calculated. Crossover temperatures are present in all these quanti ties and appear compatible with each other and with crossover temperatures extracted from the long relaxation process. From a structural point of view, the water HBs network results strongly altered by the presence of the different chemical compounds, with a major effect due to trehalose. This is probably due to the strong interaction with other components (excluded volume effects and hydrogen bonding with protein and trehalose) and to the lower concentration of DMSO investigated in this work, that is the one commonly used in cryopreserving application. Nonetheless, comparing struc tural quantities pertaining to the two solutions with different concentration of DMSO revealed noticeable changes in the HBs network, confirming its effect in altering the structure of surrounding water. From the results reported in this work, the composite DMSO-trehalose mixture appears to be useful for cryopreservation in that the combined action of both the cryopreserving agents can be exploited.