基于上海同步辐射光源的生物小角X-射线散射技术在膜蛋白结构生物学应用中关键技术的研究

Membrane proteins have essential roles in many cellular processes, such as signal transduction, cell-to-cell communication, membrane transport, and lipid and energy metabolism. The function variety of membrane proteins makes them represent the targets of more than 60% of drugs in clinical use. However, only 1-2% of the known protein structures in the Protein Data Bank (PDB) are from membrane proteins. The progress of membrane proteins related biochemistry and structural biology study is extremely slow because of their instability and tendency to aggregate when extracted from their natural lipid-bilayer environments. In present, the techniques such as crystallography, single-particle cryo-EM and NMR are most popular way to study the structural of membrane proteins. However, each of these techniques has their limitations, especially the optimization of these technologies for individual membrane proteins is relatively laborious. In this project, we proposed a membrane protein structural study platform using the synchrotron radiation facility based biological small-angle X-ray scattering (BioSAXS) beamline. The BioSAXS has several advantages in membrane protein structural study by comparison with other techniques. Although it is limited by resolution (~10-50 Å d-spacing), SAXS is not limited to the size of biomolecules, and the sample preparation and data collection can significantly avoid laborious work. Most importantly, SAXS can reveal the dynamic structural conformations in solution. Here we focused on BioSAXS technique developments in membrane protein study. The high brightness of synchrotron X-ray beam together with optimized methods for membrane proteins reconstitutions render SAXS a powerful complementary technique even when high-resolution information is available from other methods. Molecular dynamic simulation for SAXS data processing package will be developed to obtain membrane protein conformation. Further efforts will be focused on developing automated data processing software, so that the automated data collection, data analyzing and analyzed results output could be realized in the same time. Our BL19U2 BioSAXS beamline based on Shanghai Synchrotron Radiation Facility (SSRF) would provide a practical research platform for this project, and our experiences in handling biological samples could also promote the project progress. Meanwhile, several preparatory work have been carried out in reconstituting membrane proteins and in developing practical scattering data processing software, which would definitely ensure the smooth implementation of this project. In summary, it is the first time in China to develop synchrotron radiation facility based BioSAXS platform to study biological membrane proteins. This project has both international advances and domestic innovations.

膜蛋白是生物膜功能的主要承担者，具有重要生物学功能。然而当前成功解析出三维结构的蛋白中，膜蛋白所占比例仅有1-2%。常规膜蛋白结构生物学研究常采用晶体学、单颗粒冷冻电镜以及核磁共振方法，但每种方法都具有一定局限性。本项目将基于同步辐射生物小角X-射线散射技术（BioSAXS），针对现有膜蛋白结构生物学研究各方法局限性，提出膜蛋白结构研究的BioSAXS技术研发方案。通过优化膜蛋白纯化表达体系，提高样品均一性；并在此基础上发展基于散射数据的结构模拟算法获得膜蛋白的基本构象；进而开展膜蛋白研究的SAXS自动数据处理程序开发。在前期工作中，申请人已针对膜蛋白的小角散射测试特殊性进行了深入研究。掌握了不同膜蛋白构建体系、膜蛋白散射数据结构重建等关键问题的解决方案。本项目首次在BioSAXS线站上提出膜蛋白结构生物学检测平台的研发方案，对突破现有膜蛋白结构研究方法的瓶颈具有重要意义。

膜蛋白是生物膜功能的主要承担者，具有重要生物学功能。然而常规的结构生物学实验方法，如：晶体学、单颗粒冷冻电镜以及核磁共振等，在膜蛋白结构表征方面都具有一定的局限性。本项目基于同步辐射生物小角X-射线散射线站，围绕项目需要解决的关键科学问题开展了一系列研究工作。首先，通过在线整合高效液相色谱分子筛层析技术，优化复杂膜蛋白实验体系，提高样品的稳定性与均一性；进而针对现有膜蛋白结构生物学研究方法的局限性，成功在线实现了原位多检测器联用研究复杂生物膜蛋白微观结构的同步辐射散射实验平台搭建；并在此基础上发展基于散射数据的结构模拟算法，完成了溶液散射数据自动分析处理程序SAS-cam的开发。本项目首次在同步辐射装置上提出膜蛋白结构生物学检测平台的研发方案，对突破现有膜蛋白结构研究方法的瓶颈具有重要意义。在平台建设方面，成功搭建了基于同步辐射装置的生物膜蛋白散射检测平台，有效解决了X-射线散射实验方法对复杂体系均一性以及膜蛋白体系中糖类与脂类定量的技术瓶颈，为开展膜脂、膜蛋白相关的课题研究奠定了技术基础。在算法开发方面，项目团队在完成装置底层控制的基础上，自主开发了散射数据自动分析处理程序；该程序能够自动、高效、批量地分析溶液散射数据，帮助用户在实验的同时随时优化调整实验方案，极大地提高了同步辐射机时利用率。在方法应用方面，分别对结核分枝杆菌基因组编码相关的重要膜蛋白MmpL分子的聚合状态、G蛋白偶联受体（GPCR）膜蛋白家族成员分子结构、配方奶粉中重要营养元素酪蛋白结构稳定性等具有重要生物学意义的复杂膜蛋白体系微观结构进行了表征，支持了一批与重要生命现象或疾病相关的膜蛋白结构表征相关成果产出。基于本项目的资助，目前已发表一作与通讯作者论文7篇，合作作者论文5篇。出版学术译著1部。相关研究成果拟申请软件著作权1项。