Studies of the natural force-induced unfolding of Von Willebrand Factor

自然力诱导血管性血友病因子展开的研究

• 批准号: BB/E012132/1
• 负责人: Philip Williams
• 金额: $ 41.16万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE012132%2F1
• 关键词: Studies; natural; force; induced; unfolding

When a blood vessel is damaged, cells that are normally below the inner surface of the vessel are exposed to blood. The blood protein, Von Willebrand factor (VWF), then binds to exposed collagen in the vessel and undergoes a conformational change to reveal platelet binding sites. VWF (in large multimeric forms) binds several platelets, which aggregate forming a platelet plug / eventually leading to the formation of a blood clot. Within this process a second VWF cleaving protein (ADAMTS-13) is required to cleave the VWF strands, to prevent the multimers becoming too large and adhesive. Absence of this enzyme is known to lead to diseases such as purpurea. It has been shown that tensile force, caused by rapidly flowing blood helps the cleavage of VWF by ADAMTS-13, believed to be due to unfolding of VWF with consequent exposure of the cleavage site. Understanding how force unfolds VWF is therefore essential to understanding its natural biological function. In this project we propose to investigate the force-induced unfolding of the VWF system by exploiting recent developments in ultra-sensitive force measurements techniques, such as the Biomembrane Force Probe (BFP). The use of force as a denaturant has given new insight into the role of force in a range of biological processes, and as a consequence, researchers have begun to consider force as an important parameter to include in studies of biomolecular structure and function. The atomic force microscope (AFM) has been used for most of these studies, but it is fundamentally limited in this application (by factors such as cantilever stiffness and hydrodynamics) and thus different instrumentation is required to study force-induced unfolding in greater detail. The biomembrane force probe (BFP) is the only instrument capable of spanning a sufficiently positioned and large range of force loading rates, and thus able to overcome these limitations. We at Nottingham have the only BFP of its type outside of the laboratories of its pioneer, Evan Evans, and are the only people to have undertaken dynamic force spectroscopy (DFS) of a protein using a BFP. The project builds on our preliminary experimental studies that have demonstrated that the BFP can provide new insight into protein unfolding under force. Within this project an improved understanding of protein unfolding and the affect of force will emanate, leading to a greater understanding of Nature's design and evolution of unfolding/folding energy landscapes. The project will make a significant impact in our understanding of the properties of VWF in particular, but also on the folding and unfolding of proteins in general in a physiological environment of force.

当血管受损时，通常在血管内表面以下的细胞暴露于血液。血液蛋白，血管性血友病因子（VWF），然后结合到血管中暴露的胶原蛋白，并经历构象变化，以显示血小板结合位点。VWF（大的多聚体形式）结合几个血小板，其聚集形成血小板栓/最终导致血凝块的形成。在这个过程中，需要第二个VWF切割蛋白（ADAMTS-13）来切割VWF链，以防止多聚体变得太大和粘附。缺乏这种酶已知会导致疾病，如紫癜。已经表明，由快速流动的血液引起的张力有助于ADAMTS-13切割VWF，认为这是由于VWF的解折叠以及随后切割位点的暴露。因此，了解力如何展开VWF对于了解其天然生物学功能至关重要。在这个项目中，我们建议利用超灵敏力测量技术的最新发展，如生物膜力探针（BFP），研究力诱导的VWF系统展开。力作为变性剂的使用使人们对力在一系列生物过程中的作用有了新的认识，因此，研究人员已经开始考虑将力作为生物分子结构和功能研究中的一个重要参数。原子力显微镜（AFM）已被用于大多数这些研究，但它是从根本上限制在这个应用程序（由因素，如悬臂刚度和流体力学），因此需要不同的仪器来研究力诱导的展开更详细。生物膜力探针（BFP）是唯一能够跨越充分定位和大范围力加载速率的仪器，因此能够克服这些限制。我们在诺丁汉有唯一的BFP的类型以外的实验室的先驱，埃文埃文斯，是唯一的人进行动态力光谱（DFS）的蛋白质使用BFP。该项目建立在我们的初步实验研究的基础上，这些研究表明BFP可以为蛋白质在外力下的解折叠提供新的见解。在这个项目中，将产生对蛋白质展开和力的影响的更好理解，从而更好地理解自然界的设计和展开/折叠能量景观的演变。该项目将对我们特别是对VWF性质的理解产生重大影响，而且还将对蛋白质在生理力环境中的折叠和展开产生重大影响。

项目成果

The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function

• DOI: 10.1182/blood-2009-12-257949
• 发表时间: 2010-06-10
• 期刊: BLOOD
• 影响因子: 20.3
• 作者: Luken, Brenda M.;Winn, Luke Y. N.;Crawley, James T. B.
• 通讯作者: Crawley, James T. B.

Biomembrane force probe investigation of RNA dissociation.

RNA 解离的生物膜力探针研究。

• DOI: 10.1007/s00249-010-0642-7
• 发表时间: 2011
• 期刊: EBJ
• 作者: Brampton C