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

当血管受损时，通常位于血管内表面以下的细胞暴露在血液中。血液蛋白，Von Willebrand因子(VWF)，然后与血管中暴露的胶原蛋白结合，并经历构象变化，揭示血小板结合部位。VWF(以大的多聚体形式)结合多个血小板，这些血小板聚集在一起形成血小板堵塞，最终导致血栓的形成。在这个过程中，需要第二个VWF裂解蛋白(ADAMTS-13)来裂解VWF链，以防止多聚体变得太大和粘连。这种酶的缺失被认为会导致像紫斑症这样的疾病。研究表明，血液快速流动引起的拉力有助于ADAMTS-13对VWF的切割，据信这是由于VWF的展开，从而暴露了切割部位。因此，了解力量是如何展开VWF的，对于了解其自然生物学功能是至关重要的。在这个项目中，我们建议通过利用超灵敏测力技术的最新发展，如生物膜力探针(BFP)，来研究力诱导VWF系统的展开。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，研究人员开始将力作为一个重要的参数来包括在生物分子结构和功能的研究中。原子力显微镜(AFM)已经被用于这些研究的大部分，但它在这一应用中基本上是有限的(受悬臂刚度和流体动力学等因素)，因此需要不同的仪器来更详细地研究力诱导的展开。生物膜力探头(BFP)是唯一一种能够跨越足够位置和大范围的力负荷速率的仪器，因此能够克服这些限制。在诺丁汉，我们拥有先驱埃文·埃文斯实验室之外唯一的这类BFP，也是唯一使用BFP对蛋白质进行动态力光谱(DFS)的人。该项目建立在我们的初步实验研究的基础上，这些研究已经证明，BFP可以为蛋白质在力的作用下展开提供新的见解。在这个项目中，将对蛋白质展开和力的影响产生更好的理解，从而更好地理解自然对展开/折叠能量景观的设计和进化。该项目将对我们理解VWF的性质产生重大影响，而且还将对蛋白质在力的生理环境中的折叠和展开产生重大影响。

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.