Thermodynamics and kinetics of membrane protein oligomerization in bilayers

化学部的生命过程化学项目为这项工作提供了资金。肯塔基大学的魏益南教授研究了细胞膜中的蛋白质-蛋白质相互作用，这些相互作用导致了功能性蛋白质复合体的组装。选择性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通透的屏障上的门，允许物质和信号通过膜选择性地交换。了解导致膜蛋白质结构的分子识别过程对于更好地理解蛋白质的进化、功能和调控是至关重要的。这项研究的目的是建立一个模型系统，一个多药物外排转运体，AcrB，在活细胞中的络合清晰的图像，并确定它在细胞膜上的稳定性。这一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。该项目还纳入了一项向12-18岁女孩介绍科学和科学事业的外联计划。魏医生与肯塔基州Mt Sterling的希望山青年服务项目合作，该项目面向12-18岁的女孩，这些女孩有虐待和疏忽的历史，有行为和情感问题，需要广泛的治疗和治疗。希望山青年服务社没有接受STEM(科学、技术、工程和数学)教育，因此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。这些互动的目的是拓宽女孩对职业机会的了解。在本研究中，AcrB被用作模型系统，并对其在洗涤剂胶束和脂类双层中的齐聚作用进行了定量表征。利用在初步研究中开发的一系列分析和方法，在细胞和重组脂双层中监测三聚体组装过程的动力学和热力学方面。这些方法包括：基于荧光共振能量转移(FRET)的方法，该方法与脂类双层环境中AcrB亚单位相互作用的表征相兼容；基于荧光的方法，用于监测蛋白质的展开；以及基于二硫键捕获的方法，用于表征AcrB在细胞膜中的天然状态的结构。这些分析方法适用于监测和表征AcrB在洗涤剂胶束和脂质双层中的齐聚反应。此外，还创建了一组三聚体亲和力降低的AcrB突变体。1)AcrB三聚体在细胞内组装过程的研究；2)细胞膜中AcrB三聚体稳定性的测定；3)AcrB三聚体在脂质双层中解离的特征。该项目的成果为组装多跨多结构域的膜蛋白三聚体的动力学和热力学带来了新的见解。研究中开发的方案和参数为研究界提供了有价值的工具和基准，从而引领了膜蛋白研究的新举措。

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.