Thermodynamics and kinetics of membrane protein oligomerization in bilayers

化学部的生命过程化学项目资助了这项工作。来自肯塔基州大学的Yinan Wei教授研究了细胞膜中蛋白质与蛋白质之间的相互作用，这些相互作用导致功能性蛋白质复合物的组装。 选择性渗透膜定义了单个细胞和细胞器的边界，对于维持生命的能量生产至关重要。膜蛋白是这些高度不可渗透屏障上的门，允许跨膜的物质和信号的选择性交换。理解导致膜蛋白结构的分子识别过程对于提高对蛋白质进化、功能和调节的理解是必不可少的。本研究的目的是建立一个清晰的图片的一个模型系统，多药外排转运蛋白，AcrB，在活细胞中的复合，并确定其在细胞膜的稳定性。这种追求使研究生和本科生能够获得生物化学和分子生物学研究方面的培训。该项目还纳入了一个外联方案，向12至18岁的女孩介绍科学和科学职业。魏博士与希望山青年服务（Mt Sterling，KY）合作，这是一个针对12-18岁女孩的项目，这些女孩有虐待和忽视的历史，并且有需要广泛治疗和治疗的行为和情感问题。希望山青年服务没有STEM（科学，技术，工程和数学）教育，所以他们的学生在数学和科学方面落后;女孩们努力理解教育可以为她们提供的机会，缺乏梦想和动力。这些互动的目的是扩大女孩对职业机会的了解。 在这项研究中，AcrB被用作一个模型系统和它的低聚在洗涤剂胶束和脂质双层定量的特点。三聚体组装过程的动力学和热力学方面的监测在细胞和重构的脂质双层使用一系列的测定和方法开发的初步研究。其中包括：一种基于荧光共振能量转移（FRET）的方法，该方法与脂双层环境中AcrB亚基相互作用的表征相容，一种基于荧光的方法来监测蛋白质解折叠，以及一种基于二硫键捕获的方法来表征细胞膜中AcrB在其天然状态下的结构。这些测定适合于监测和表征洗涤剂胶束和脂质双层中的AcrB寡聚化。此外，已经产生了具有降低的三聚体亲和力的AcrB突变体的集合。本论文的主要研究内容如下：1）研究AcrB三聚体在细胞中的组装过程; 2）测定AcrB三聚体在细胞膜中的稳定性; 3）表征AcrB三聚体在脂双层中的解离。该项目的成果为多跨度和多结构域膜蛋白三聚体组装的动力学和热力学带来了新的见解。在研究中开发的协议和参数为研究界提供了有价值的工具和基准，这为膜蛋白研究的新举措开辟了道路。

The Chemistry of Life Processes Program in the Chemistry Division funds this work. Professor Yinan Wei from University of Kentucky investigates protein-protein interactions in the cell membrane that lead to the assembly of functional protein complexes. Selective-permeable membranes, which define the boundaries of individual cells and cellular organelles, are essential for energy production to sustain life. Membrane proteins are gates on these highly impermeable barriers, allowing selective exchange of substances and signals across the membrane. Understanding of the molecular recognition process leading to membrane protein structures is essential for improved understanding of protein evolution, function, and regulation. The aim this research is to establish a clear picture of the complexation of one model system, a multidrug efflux transporter, AcrB, in living cells and determine its stability in the cell membrane. This pursuit allows graduate and undergraduate students to acquire training in biochemistry and molecular biology research. This project also integrates into an outreach program to introduce science and science careers to girls ages 12-18. Dr. Wei is collaborates with the Hope Hill Youth Service (Mt Sterling, KY), a program for girls ages 12-18, who suffer from histories of abuse and neglect and have behavioral and emotional problems requiring extensive treatment and therapy. The Hope Hill Youth Service does not have STEM (Science, Technology, Engineering and Mathematics) education and so their students fall behind in math and science; the girls struggle to understand the opportunities that education can afford them, lacking dreams and motivation. The goals of these interactions are to broaden the knowledge of career opportunities for the girls.  In this study, AcrB is used as a model system and its oligomerization in both detergent micelles and lipid bilayers is quantitatively characterized. The kinetic and thermodynamic aspects of the trimer assembly process are monitored in cells and in reconstituted lipid bilayers using an array of assays and methods developed in preliminary studies. These include: a fluorescence resonance energy transfer (FRET)-based method that is compatible with the characterization of AcrB subunit interaction in a lipid bilayer environment, a fluorescence-based method to monitor protein unfolding, and a disulfide-trapping based method to characterize the structure of AcrB in the cell membrane in its native state. These assays are suitable for monitoring and characterizing AcrB oligomerization in both detergent micelles and lipid bilayers. Furthermore, a collection of AcrB mutants with decreased trimer affinities have been created. The following research objectives are pursued: 1) investigation of the process of AcrB trimer assembly in cells; 2) determination of AcrB trimer stability in the cell membrane; and 3) characterization of AcrB trimer dissociation in lipid bilayers. Outcomes from this project bring new insight into the kinetics and thermodynamics of assembly of a multi-span and multi-domain membrane protein trimer. The protocols and parameters developed in the research provide valuable tools and benchmarks for the research community, which lead the way to new initiatives in membrane protein research.