分析超速离心技术研究膜蛋白TmrAB  

Study of Membrane Protein TmrAB by Analytical Ultracentrifugation

作　　者：褚文丹 徐扬 周翠燕 芦亚菲 于晓霞 张瑞轩 李文奇 CHU Wen-Dan;XU Yang;ZHOU Cui-Yan;LU Ya-Fei;YU Xiao-Xia;ZHANG Rui-Xuan;LI Wen-Qi(School of Biomedicine in Tsinghua University,Notional Protein Science Facility,Beijing 100084;School of Life Science,Tsinghua University,Beijing 100084;Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101;Corresponding author)

机构地区：[1]清华大学生命科学与医学研究院蛋白质研究技术中心,北京100084 [2]清华大学生命科学学院,北京100084 [3]中国科学院生物物理研究所,北京100101

出　　处：《生物化学与生物物理进展》2018年第10期1047-1053,共7页Progress In Biochemistry and Biophysics

基　　金：中国博士后科学基金面上资助(2014M550050);清华大学结构生物学高精尖创新中心资助项目~~

摘　　要：去垢剂在膜蛋白的提取纯化过程中起到必要的作用,对膜蛋白的聚合状态、结晶条件以及理化性质等方面都有较大影响.分析超速离心技术(analytical ultracentrifuge,AUC)通过测定溶液中膜蛋白-去垢剂复合物在离心场中的沉降运动轨迹,可以分析获得其沉降系数、摩尔质量、流体力学半径、结合常数等水力学和热力学性质,进而判断膜蛋白-去垢剂复合物的均一性及聚合状态.本文以嗜热菌来源的ATP结合转运蛋白(ABC transporter)TmrAB作为研究对象,利用分析超速离心技术结合分子排阻层析和冷冻电镜负染技术,研究其均一性、聚合状态以及去垢剂与膜蛋白的摩尔比.结果显示,在8倍临界胶束浓度(critical micelle concentration,CMC)的DDM条件下,TmrAB性质均一,并以异二聚体的单体形式存在,DDM与Tmr AB的摩尔比为116∶1.本研究表明,分析超速离心技术是一种测定膜蛋白分子质量、研究膜蛋白聚合状态的可靠手段.Detergent is critical for membrane protein purification, impacting the state of oligomerization, crystallization conditions and other physicochemical properties. Analytical ultracentrifugation （AUC）, by characterizing the sedimentation of membrane protein-detergent complex in centrifugal fields, is able to measure various hydrodynamic and thermodynamic properties, including sedimentation coefficient, molar mass, hydrodynamic radius, binding coefficient, thus defining the protein-detergent complex. This study focuses on an ABC homogeneity and oligomerization state of membrane transporter from thermophilic bacteria, TmrAB, and utilizes AUC coupled to size-exclusion chromatography and negative staining electron microscopy to determine its homogeneity, oligomerization, and stoichiometry of membrane protein and detergent molecules. The results indicate that TmrAB complex exists as homogeneous monomer of heterodimers of TmrA and TmrB, in the condition of 8 ~ Critical Micelle Concentration （CMC） DDM, having a ratio of DDM/TmrAB equal to 116 ： 1. This study suggests, AUC is a reliable method to analyze the molecular mass of membrane protein.

关 键 词：分析超速离心 分子排阻层析 冷冻电镜 膜蛋白 分子质量 

分 类 号：Q5[生物学—生物化学]