Novel Methodologies for the Preservation (and Recovery) of Proteins at Low Temperature

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ABSTRACT The goal of this SC3 grant is to develop a novel method for the cryopreservation of proteins without the use of co-solvents, which are usually employed to limit the structural changes that can occur during freezing. The method exploits the glass polymorphism of water, i.e., low density amorphous (LDA) and high density amorphous (HDA) glasses can be produced following specific cooling/compression pathways. The pathways for storage and recovery of proteins will be studied using non-equilibrium molecular dynamic simulations coupled to all-atom force fields. The central hypothesis is that the density of the HDA will limit fluctuations in the solution upon cooling and the protein will not undergo any significant structural changes when vitrified in HDA. The rationale for the proposed research is that an understanding of the detailed mechanism by which proteins are preserved in glassy water, can lead to the rational design of experiments that allow for more controlled subzero protein preservation. In order to systematically develop this understanding three groups of systems, with increasing order of structural complexity will be studied: (i) short peptides with different secondary structures (ii) simple proteins that contain these secondary structures within their tertiary structures, and (iii) two biomedically relevant proteins. The information obtained from these studies has the potential to be extended to the storage of pharmaceuticals, food, and biological cells without the use of co-solvents.