SHEAR INDUCED DENATURATION OF PROTEINS

剪切引起的蛋白质变性

• 批准号: EP/F007736/1
• 负责人: Stavroula Balabani
• 金额: $ 38.61万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FF007736%2F1
• 关键词: SHEAR; INDUCED; DENATURATION; PROTEINS

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.

蛋白质在根本上是对生命至关重要的重要分子，现在广泛用于工业和医疗应用中。仅蛋白质药物行业的价值每年价值3亿美元，并且正在迅速增长。蛋白质是高度复杂的聚合物，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已被长期认可，并导致了数十年来研究蛋白质的研究（或蛋白质变性），包括医疗应用在内的旋转（包括医疗蛋白质）（未折叠的蛋白质可以导致阿尔茨海默氏病和诸如阿尔茨海默氏症的致命疾病，例如阿尔茨海默氏症和帕金森氏症）和科学技术（蛋白质固定的工具和化学工具），并在化学上使用了整体化学和化学含量，并提供了整体化的工具和化学含量。是深入研究的领域，现在机械发展被用作纳米型工程的新工具，实际上，对于剪切力在液体中展开的是什么都不知道。任何用于控制蛋白质结构和展开的新工具都将是一个重大突破，并且有可能使用液体（大多数蛋白质的自然环境和工业中蛋白质制备的大多数阶段）进行此操作，这使得剪切流是令人难以置信的有希望的工具。但是，我们必须发现管理这一现象的自然法律，并开发实际的工具来测量和控制剪切引起的展开，然后才能使用它。我们将对剪切流对蛋白质结构的影响进行最全面的研究，涵盖了不同形状和稳定性的蛋白质范围，从而使实验性和溶液的影响更加多种多样，并更加敏感。我们的目的不仅是在剪切下确定哪些蛋白质会发生或不进行展开，而且要识别和量化蛋白质结构的哪些部分变化（这在生理上比说蛋白质确实或不展开更重要），还要了解剪切诱导的变性如何发生并开发出控制和操纵蛋白质的方法。这项研究还将涉及与蛋白质相关的层流和剪切流参数的首次全面分析，这是获得对过程的真实机械理解所需的。首先，我们将表征和量化要使用的流量单元的剪切电池的剪切参数（宏观和微富集设备），然后从范围内识别较大的蛋白质，而不是在范围内识别范围或不及格。不同的蛋白质可能具有巨大不同的结构和稳定性，并且在我们的实验条件下可能不会展开某些蛋白质，有些蛋白质会展开，而另一些蛋白质可能需要通过控制实验参数（pH，粘度等）来展开。 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通过控制溶液特性（流速，粘度，pH，化学添加剂）来展开的Manipultae蛋白。通过这种方式，我们将了解哪种类型的蛋白质最容易受到剪切流的影响，以及为什么，我们将开发工具和技术来控制剪切诱导的变性，从而使其成为蛋白质工程工具包的新补充。

项目成果

Susceptibility of different proteins to flow-induced conformational changes monitored with Raman spectroscopy.

• DOI: 10.1016/j.bpj.2009.10.010
• 发表时间: 2010-02
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: L. Ashton;J. Dusting;Eboshogwe Imomoh;S. Balabani;E. Blanch