Biophysics of cryopreservation: elucidating the structural architecture and physical mechanisms of both model and complex biological systems

许多生活在极端环境中的生物已经发展出保护它们免受低温等环境压力的机制。冷冻保存是一种有效的过程，其中细胞或整个组织通过冷却到零度以下的温度来保存。冷冻保护剂分子甘油在生命系统中普遍存在，在稳定生物体对抗不利环境条件方面起着至关重要的作用。甘油作为冷冻保护剂的作用很可能与甘油、生物体和大量水环境之间的相互作用有关。了解甘油/水系统本身的分子相互作用是了解甘油溶液中更复杂系统的必要的第一步。因此，揭示冷冻保存的全部潜力在很大程度上依赖于提高我们对所涉及的物理相互作用和分子机制的理解。这些机制决定了感兴趣的系统的结构架构，并最终决定了随后的生物和化学反应的动力学。采用结构方法首先确定简单系统（如含水冷冻保护剂系统）中冷冻保存的原理，将提供初始分子模型。将这些模型扩展到真实的生物系统，例如冷冻保护剂环境中的蛋白质折叠，将提供测试、改进和开发这些模型的手段。开始，我将探索一个简单的冷冻保护剂系统，水甘油，使用一种实验技术称为中子衍射。该技术提供了系统结构特性的信息。接下来，我将探索冷冻保护剂在更复杂系统中的物理机制。我将研究在不同的冷冻保护剂环境中个别蛋白质的折叠和展开特性。我将使用的实验技术是单分子力钳光谱学。在力钳光谱中，单个蛋白质分子被保持在恒定的拉伸力下，使得可以观察到解折叠和重折叠过程作为时间的函数。我将在利兹大学开发和建造一个定制的力钳仪器。我将使用这个仪器在恒定的力下机械地展开单个蛋白质，并检查蛋白质展开的物理性质。

Many organisms that live in extreme environments have developed mechanisms that protect them from environmental stresses such as low temperatures. Cryopreservation is an effective process where cells or whole tissues are preserved by cooling to sub-zero temperatures. The cryoprotectant molecule glycerol is ubiquitous in living systems where it plays a vital role in stabilizing organisms against adverse environmental conditions. Glycerol's role as a cryoprotectant is most likely linked to the interactions between glycerol, the organism and bulk water environment. Understanding the molecular interactions of the glycerol/water system itself is a necessary first step toward understanding higher complexity systems in glycerol solutions. Uncovering the full potential of cryopreservation therefore heavily relies on improving our understanding of the physical interactions and molecular mechanisms involved. These mechanisms determine the structural architecture of the system of interest and ultimately the dynamics of subsequent biological and chemical reactions. Taking a structural approach to first determine the principles of cryopreservation in simple systems such as an aqueous cryoprotectant system, will provide initial molecular models. Extending these models to real biological systems, such as protein folding in cryoprotectant environments will provide a means to test, refine and develop these models. To begin with, I will explore a simple cryoprotectant system, aqueous glycerol, using an experimental technique called neutron diffraction. This technique provides information on the structural properties of the system. Next, I will explore the physical mechanisms of cryoprotectants in a more complex system. I will examine the folding and unfolding properties of individual proteins in different cryoprotectant environments. The experimental technique I will utilize is single molecule force-clamp spectroscopy. In force-clamp spectroscopy a single protein molecule is held at a constant stretching force, such that the unfolding and refolding processes can be observed as a function of time. I will develop and construct a custom-built force-clamp instrument at the University of Leeds. I will use this instrument to mechanically unfold single proteins at a constant force and examine the physical properties of protein unfolding.