SHEAR INDUCED DENATURATION OF PROTEINS

蛋白质是对生命至关重要的重要分子，现在正广泛应用于工业和医疗领域。仅蛋白质药物行业每年就价值3000亿美元，而且还在快速增长。蛋白质是高度复杂的聚合物，它们必须折叠成正确的结构才能有效发挥作用。这并不简单，蛋白质折叠的重要性早已被认识到，并导致了数十年对蛋白质展开(或蛋白质变性)的研究，其副产品包括医疗应用(未折叠的蛋白质可导致阿尔茨海默氏症和帕金森氏症等致命疾病)和科学技术(蛋白质展开工具经常用于生物学和化学)。尽管通过化学和热方法展开蛋白质是深入研究的领域，现在机械展开正被用作纳米生物工程的一种新工具，但几乎对流体中剪切力展开知之甚少。任何控制蛋白质结构和展开的新工具都将是一项重大突破，使用流体(工业中大多数蛋白质和蛋白质制备的大多数阶段的自然环境)来实现这一点的可能性，使剪切流成为一种非常有前途的工具。然而，我们必须发现控制这一现象的自然规律，并开发实用的工具来测量和控制剪切诱导展开，然后才能利用它。我们将对剪切流对蛋白质结构的影响进行迄今尝试过的最全面的检测，涵盖不同形状和稳定性的各种蛋白质，调查实验条件和溶剂性质的影响，并使用比以前带来的更敏感的工具来解决这个问题。我们的目标不仅是识别哪些蛋白质在剪切下发生或不发生展开，而且识别和量化蛋白质结构的哪些部分发生变化(这比仅仅说蛋白质发生或不展开更具生理学意义)，了解剪切诱导变性如何发生的机制，并开发控制和操纵蛋白质展开的方法。这项研究还将首先对与蛋白质有关的层流和剪切流动参数进行全面分析，这是获得对该过程的真正机理理解所必需的。首先，我们将表征和量化将要使用的流动细胞(宏观和微观流体装置)的剪切参数，然后从一组变化很大的目标中确定哪些蛋白质在流体流动中展开或不展开。不同的蛋白质可能具有非常不同的结构和稳定性，在我们的实验条件下，有些蛋白质可能不会展开，有些会展开，而另一些可能需要通过控制实验参数(pH、粘度等)来帮助展开。重要的是要研究一些具有不同形状和内在稳定性的非常不同的蛋白质，以确定一般趋势或规律，并为项目第二阶段确定有利的目标。然后，我们将对那些被发现在剪切下展开的蛋白质进行更深入的研究，目的是确定蛋白质结构的哪些部分发生变化(这比仅仅知道蛋白质是否展开更重要)，量化这些变化，详细说明相关机制，并学习如何通过控制溶液特性(流量、粘度、pH值、化学添加剂)来操纵蛋白质展开。通过这种方式，我们将了解哪些类型的蛋白质最容易受到剪切流动的影响，以及为什么，我们将开发控制剪切诱导变性的工具和技术，使其成为蛋白质工程工具包中的一个新成员。

Proteins are fundamentally important molecules crucial for life and are now becoming widely used in industrial and medical applications. The protein drug industry alone is worth $US300billion per year and is growing quickly. Proteins are highly complex polymers and they have to fold into their correct structures to function effectively. This is not simple and the importance of protein folding has been long recognised and has led to decades of research into protein unfolding (or protein denaturation), with spin offs including medical applications (unfolded proteins can cause deadly diseases such as Alzheimer's and Parkinson's) and scientific technologies (protein unfolding tools are routinely used in biology and chemistry).Although protein unfolding by chemical and thermal means are areas of intensive research, and now mechanical unfolding is being utilised as a new tool in nanobioengineering, virtually nothing is known about unfolding by shear forces in fluids. Any new tool for controlling protein structure and unfolding will be a major breakthrough and the possibility of doing this using fluids (the natural environment for most proteins and most stages of protein preparation in industry), makes shear-flow an incredibly promising tool. However, we must discover the natural laws governing this phenomenon and develop the practical tools to measure and control shear-induced unfolding before we can make use of it.We will conduct the most comprehensive examination of the effects of shear flow on protein structure yet attempted, covering a diverse range of proteins of different shapes and stabilities, investigate the effects of experimental conditions and solvent properties, and use far more sensitive tools than have previously been brought to this problem. Our aim is to not only identify which proteins do or do not undergo unfolding under shear, but to identify and quantify which parts of the protein structure change (this is more physiologically important than just saying a protein does or doesn't unfold), learn the mechanisms of how shear-induced denaturation occurs and develop the methods to control and manipulate protein unfolding. This study will also involve the first comprehensive analysis of laminar and shear flow parameters in relation to proteins, which is required to obtain a true mechanistic understanding of the process.First, we will characterise and quantify the shear parameters of the flow cells to be used (both macro- and micro-fluidic devices) and then identify which proteins, from a widely varied set of targets, do or do not unfold in fluid flows. Different proteins can have greatly different structures and stabilities and it is likely that some proteins will not unfold under our experimental conditions, some will unfold, while others may need assistance to unfold by controlling experimental parameters (pH, viscosity etc.). It will be important to examine a number of very different proteins with different shapes and inherent stabilities to identify general trends or rules, and to identify favourable targets for the second phase of the project.We will then conduct more intensive studies for those proteins found to unfold under shear, with the aim of determining which parts of the protein structure change (which is more important than just knowing if the protein unfolds or not), quantifying these changes, detailing the mechanisms responsible and learning how to manipultae protein unfolding by controlling the solution characteristics (flow rate, viscosity, pH, chemical additives). In this way we will learn which types of proteins are most susceptible to shear flows and why, and we will develop the tools and techniques to control shear-induced denaturation, making it a new addition to the protein engineering toolkit.