基于原子力显微镜定量分析单个凝血蛋白在生物膜表面的取向与功能

项目名称： 基于原子力显微镜定量分析单个凝血蛋白在生物膜表面的取向与功能

项目编号： No.11474298

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 数理科学和化学

项目作者： 吕军鸿

作者单位： 中国科学院上海应用物理研究所

项目金额： 100万元

中文摘要： 凝血蛋白八因子(FVIII)是处于凝血反应中心位置的一种外周膜蛋白，其功能与血栓和心血管疾病密切相关。我们前期研究发现与生物膜结合的氨基酸以及结合其膜界面结构域的抗体、膜物理化学性质都可明显调控FVIII的活性，提示FVIII的功能很可能与其在膜上的取向有关。常规研究蛋白取向的技术很难用于膜结合蛋白，并且多是群体水平测量，更不能动态研究膜物理性质与蛋白质功能之间的关系。本项目旨在发展基于原子力显微镜(AFM)对膜表面的单个蛋白质分子高度精确测量的技术，直接观察和定量分析FVIII在膜表面的取向，然后结合课题组最近建立的AFM纳米力学成像和非接触高分辨成像新技术，研究生物膜的相变、电荷和曲率等物化性质以及蛋白质的膜结合界面及其抗体对取向的影响，从而在单分子水平上为研究FVIII等外周膜蛋白在膜表面的结合与取向提供试验方法和证据支持，为揭示生物膜的物理性质对膜蛋白功能调控的机制奠定基础。

中文关键词： 原子力显微镜；膜蛋白；界面取向；凝血蛋白；生物膜

英文摘要： Coagulation protein factor VIII (FVIII), one of key peripheral membrane proteins in blood coagulation, is associated with thrombus and cardiovascular disease. Our previous experimental results have shown that the activity of FVIII is influenced by site-directed mutagenesis of the amino acids on the membrane-interactive surface as well as the antibodies against these interfaces, and also regulated by the lipid membrane, indicating the full activity of FVIII may be related to its right orientation on a membrane surface. However, current available techniques for protein orientation on solid surfaces are not suitable for these kind of membrane-bound proteins because of the dynamic nature of lipid bilayers and can merely provide the ensemble averaging information based on bulk measurements. This proposal aims at developing an atomic force microscopy（AFM）-based technique to quantitatively determine the orientation of proteins on a membrane surface through the height measurement of single protein molecules. Combined with nanomechanical imaging and non-contact high-resolution imaging techniques of AFM constructed recently in our group, this developing method will be used to explore the orientation of FVIII on the membrane surface by investigating the role of the amino acids in and antibodies against its membrane-interactive domains. The effect of the physical properties of cell membranes, such as phase separation, surface charge, and membrane curvature, on the protein orientation will be also investigated. These results obtained would not only provide experimental methodology and direct evidence on the binding and orientation of membrane-bound proteins, such as FVIII, but also be of great interest in revealing the regulation relationship of the protein function within or upon a membrane surface.

英文关键词： Atomic force microscopy;membrane protein;orientation;coagulation protein;lipid bilayer