BRIGE: A Research and Education Program for the Development of Technology for Transmembrane Proteomics

EEC-0824381丹尼尔这项布里奇提案的总体目标是启动一项研究和教育计划，以开发用于快速评估膜组成结构和功能的新技术。通过将膜生物物理、化学和工程方面的技术专长与广泛的教育目标和推广工作相结合，该项目将促进我们对支持的脂质双分子层中电泳分离的物理-化学影响的基本理解，同时为学生作为科学家和工程师的职业生涯做好准备，并欣赏并致力于多样性。研究的目标是开发和了解双层电泳法分离膜物种的机理，从而创造出将细胞物种分离与芯片上各种单元操作相结合的设备。假设膜平台中的生物分子分离可以通过三种机制发生：1)在双层化学或温度的促进下，双分子层和主体水相内外的组分的差异分配；2)一种物种优先分配到脂筏中，而另一种物种留在脂筏外；以及3)由流体双层中的障碍引起的流体动力学效应。从这些研究中获得的洞察力将被用于优化更复杂的物种的分离，即外周蛋白，并最终分离跨膜物种。方法是配制不同组成和性质的双层，然后测量其中膜组件的扩散、迁移率和分离效率。之前的研究表明，减少流动性会导致更好的分离。因此，可以通过添加甾醇或降低温度、组分与脂筏的相互作用以及添加不同大小和浓度的流体动力学障碍来减少双层中的扩散，从而加强分离，因为所有这些都已被证明降低了膜物种的流动性。目前尚不清楚对这些新战略的评价和分离机制的细节，这对于优化分离解决方案是必要的。涂有双层的微流控通道内的分离将与2D蛋白质结晶和化学相互作用分析相结合。这个原型装置将是未来在类似天然环境中对蛋白质和脂肪进行结构功能研究的垫脚石。拟议活动的更广泛影响是，它有可能通过创建一个仿生平台来改变膜蛋白质组学的最新水平，以快速筛选膜蛋白和脂类化学活性、膜蛋白晶体形成和结构确定，以便了解生物功能和简化药物开发。教育目标是通过发展外联计划和课堂创新，创造出使科学和工程对代表不足的群体更具包容性的方法。为了实现这一目标，将在康奈尔大学的两个多样性项目中开发和实施互动实验室项目：Catalyst(科学和工程夏令营)和多样性和未来女性周末，这两个项目都是针对高中生的。在这些活动期间，将在研究小组的充分参与下，积极招募工程学专业的学生并对学生进行指导。为了留住更多的工程学学生，材料和能量平衡核心课程将进行现代化改造，以反映当前的教学和学习方式，并纳入基于网络的互动工具，以增加所有学生的学习。研究和教育计划被仔细地结合在一起，从而增加了工程领域的多样性，这种多样性为学生、研究成果和社会带来了价值。

EEC-0824381DanielThe overall objective of this BRIGE proposal is to initiate a research and education program to develop new technologies for rapid assessment of membrane constituent structure and function. By combining technical expertise in membrane biophysics, chemistry, and engineering with broad educational goals and outreach efforts, this program is poised to advance our fundamental understanding of physical-chemical effects on electrophoretic separations in supported lipid bilayers while preparing students for careers as scientists and engineers with an appreciation for and commitment to diversity. The research objective is to develop and understand the mechanisms of separation of membrane species by bilayer electrophoresis and then to create devices that integrate separation of cellular species with various unit operations on chip. The hypothesis is that biomolecular separations in membrane platforms can occur by three mechanisms: 1) differential partitioning of the components in and out of the bilayer and bulk aqueous phase, facilitated by bilayer chemistry or temperature, 2) preferential partitioning of one species into lipid rafts while the other stays outside, and 3) by hydrodynamic effects caused by obstacles in an otherwise fluid bilayer. Insight gained from these studies will be used to optimize separations of more complex species i.e., peripheral proteins, and eventually, transmembrane species. The approach is to formulate bilayers of various compositions and properties then measure the diffusion, mobility, and separation efficiency of membrane components within it. Previous studies showed that reducing mobility leads to better separations. Therefore, separations could be enhanced by reducing diffusion in the bilayer by adding sterols or reducing temperature, component interactions with lipid rafts, and adding hydrodynamic obstacles of various sizes and concentrations, as all of these have been shown to reduce mobility of membrane species. An evaluation of these new strategies and the details of the mechanisms of separation are currently unknown and are necessary to optimize separation resolution. Separations inside microfluidic channels coated with bilayers will be integrated with 2D protein crystallization and chemical interaction assays. This prototype device will be a stepping stone towards future structure-function studies of proteins and lipids in a native-like environment. The broader impact of the proposed activities is its potential to transform the state-of-the-art in membrane proteomics by creating a biomimetic platform for rapid screening of membrane protein and lipid chemical activity, membrane protein crystal formation, and structure determination in order to understand biological function and streamline drug development. The educational objective is to create ways to make science and engineering more inclusive to underrepresented groups through development of outreach programs and innovation in the classroom. To achieve this goal, interactive laboratory projects will be developed and implemented during two of Cornell's diversity programs: CATALYST (a summer camp in science and engineering) and the Diversity and Prospective Women's Weekends, both programs targeting high school students. Active recruitment to engineering and mentoring of students will occur during these events with full participation of the research group. In an effort to retain more students in engineering, the Materials and Energy Balances core course will be modernized to reflect current teaching and learning styles and incorporate interactive web-based tools to increase the learning of all students. The research and education plan are integrated carefully so that diversity within engineering is increased and this diversity brings value the students, the research effort, and to society.