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

摘要 SC3基金的目标是开发一种新的蛋白质冷冻保存方法。 不使用共溶剂，通常用来限制结构变化，可以 发生在冷冻过程中。该方法利用了水的玻璃态，即低密度 非晶态(LDA)和高密度非晶态(HDA)玻璃可在下列特定条件下生产 冷却/压缩路径。将研究蛋白质的储存和回收途径。 使用非平衡分子动力学模拟耦合到全原子力场。中环 假设HDA的密度将限制溶液在冷却时的波动，并且 当蛋白质在HDA中玻璃化时，不会发生任何显著的结构变化。其基本原理是 因为这项拟议的研究是对蛋白质 保存在玻璃水中，可以导致合理的实验设计，允许更多 控制在零度以下的蛋白质保存。为了系统地发展这一认识 将研究三组系统，按结构复杂性的顺序递增：(I)短 具有不同二级结构的多肽(II)含有这些二级结构的简单蛋白质 它们的三级结构中的结构，以及(Iii)两种生物医学上相关的蛋白质。这个 从这些研究中获得的信息有可能扩展到存储 不使用共溶剂的药品、食品和生物细胞。

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.