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形成了保护自己免受低温等环境压力影响的机制。超低温保存是一种有效的过程，通过冷却到零度以下的温度来保存细胞或整个组织。低温保护剂分子甘油普遍存在于生命系统中，在生物抵御恶劣环境条件的过程中发挥着至关重要的作用。甘油作为低温保护剂的作用很可能与甘油、生物体和散装水环境之间的相互作用有关。了解甘油/水体系本身的分子相互作用是了解甘油溶液中更高复杂性体系的必要的第一步。因此，发现冷冻保存的全部潜力在很大程度上取决于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中的低温保存原理，例如水溶液低温保护剂系统，将提供初步的分子模型。将这些模型扩展到真实的生物系统，如在低温保护剂环境中的蛋白质折叠，将提供一种测试、改进和开发这些模型的手段。首先，我将使用一种名为中子衍射的实验技术，探索一种简单的低温保护剂系统--甘油水溶液。该技术提供了有关系统结构属性的信息。接下来，我将在一个更复杂的系统中探索低温保护剂的物理机制。我将研究单个蛋白质在不同低温保护剂环境中的折叠和去折叠特性。我将使用的实验技术是单分子力钳光谱学。在力钳光谱学中，单个蛋白质分子被保持在恒定的拉伸力，这样就可以观察到作为时间函数的去折叠和重折叠过程。我将在利兹大学开发和建造一个定制的力夹具。我将使用这台仪器以恒定的力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.