SGER: Proteomics-level Structural Biology of Transmembrane Proteins

智力价值：许多重要的细胞活动，包括离子运输、细胞间通讯、小泡运输、维持细胞结构和宿主与病原体的相互作用，都涉及嵌入细胞膜的蛋白质。跨膜(TM)蛋白质占已知蛋白质的25%以上，是目前使用的大多数药物的靶标。TM蛋白的不正确折叠和/或活性可导致正常细胞过程中的严重故障。尽管TM蛋白具有巨大的重要性，但人们对其结构和分子机制的了解远远少于对可溶性蛋白的了解。疏水序列的存在会使大量这些蛋白质的表达和分离变得困难，并使它们对许多生化和结构方法难以奏效。这个小型探索性研究(SGER)项目解决了一个普遍的问题：为什么重组蛋白表达方法导致一些TM蛋白的过度表达和正确的膜定位，而另一些却没有？更具体地说，TM蛋白的高水平表达是否存在普遍的物理障碍，或者问题更多地是由于对个别蛋白的调控？改变生长条件是否能改善TM蛋白的表达和膜的定位？一种系统的蛋白质组学方法正在被用来测试30种跨膜蛋白的表达和膜定位，这些跨膜蛋白具有不同的特征和在不同的生长条件下。研究目标包括：(A)从预测的跨膜螺旋数目、功能和其他特性不同的模型细菌系统中选择30个目标蛋白，(B)构建编码每个目标蛋白的表达载体，以确定哪些蛋白质特征影响蛋白质的表达，哪些蛋白质特征影响蛋白质在膜上的定位。这项SGER项目正在用30种蛋白质进行概念验证，然后根据结果，随后将用更多的蛋白质来进行标准建议。这个项目的成功完成可能会产生很大的影响，因为它解决了获得足够数量的TM蛋白质以研究结构和分子机制的改进方法的需要。虽然第一个项目解决了关于跨膜蛋白表达的具体问题，但在一组通用载体中使用30个基因也为未来更大规模的系统研究奠定了基础，以开发改进的TM蛋白表达方法，以及对这类重要蛋白的溶解、纯化和表征。广泛影响：这个SGER项目的更广泛影响包括在实验室培训和指导学生，以及PI的教学和推广。UIC是一所城市大学，其学生构成反映了社区，包括许多来自中等收入家庭的学生，许多家庭中第一个上大学的学生，以及来自代表性不足的群体的学生。计划和持续的活动包括：通过提供生物信息学软件、重组DNA技术、蛋白质生物化学和其他重要技术的使用经验，培训和指导生物、物理和生物工程专业的不同本科生和研究生；通过新的讲座修改和更新本科生和研究生的讲课课程，将PI的蛋白质结构和功能背景带到生物系；为大一新生开发一个新的为期一学期的部分，提供新的教材和实践经验，帮助鼓励学生考虑从事科学事业。计划的其他外展和教学活动包括在当地K-12学校(主要是少数族裔)举行的科学评审会和ACA暑期学校的嘉宾研讨会。

Intellectual Merit: Many vital cellular activities, including ion transport, cell-cell communication, vesicle transport, maintenance of cellular structure, and host-pathogen interactions, involve proteins that are embedded in the cell membrane. Transmembrane (TM) proteins make up over 25% of known proteins and are the targets for the majority of pharmaceuticals in use today. The improper folding and/or activity of TM proteins can cause severe malfunction in normal cellular processes. In spite of the vast importance of TM proteins, there is far less known about their structures and molecular mechanisms than for soluble proteins. The presence of hydrophobic sequences can make it difficult to express and isolate large amounts of these proteins and makes them refractory to many biochemical and structural methods. This Small Grant for Exploratory Research (SGER) project addresses the general question: Why do recombinant protein expression methods result in the overexpression and correct membrane localization of some TM proteins but not others? More specifically, is there a general physical barrier to high levels of TM protein expression, or is the problem due more to regulation of individual proteins? Do changing growth conditions improve TM protein expression and membrane localization? A systematic proteomics approach is being used to test the expression and membrane localization of 30 transmembrane proteins with a variety of characteristics and under a variety of growth conditions. Research aims include: (A) Selecting 30 target proteins from a model bacterial system that varies in the number of predicted transmembrane helices, function, and other characteristics and (B) Constructing expression vectors encoding each target protein to determine which protein characteristics affect protein expression and which protein characteristics affect protein localization to membranes. This SGER project is being performed with 30 proteins to test the proof of concept and then, depending upon the results, a standard proposal will follow with a larger number of proteins. The successful completion of this project is potentially high impact because it addresses the need for improved methods for obtaining sufficient amounts of TM proteins for studies of structure and molecular mechanisms. Although this first project addresses specific questions about transmembrane protein expression, use of the set of 30 genes in a common set of vectors also lays the groundwork for future larger systematic studies to develop improved expression methods for TM proteins, as well as solubilization, purification, and characterization of this important class of proteins.Broader Impacts: The broader impact of this SGER project includes training and mentoring students in the lab and teaching and outreach by the PI. UIC is an urban university whose student makeup reflects the community, including many students who are from families with modest incomes, many students who are the first in their families to go to college, and students from under-represented groups. Planned and continuing activities include: training and mentoring a diverse group of undergraduate and graduate students from the biology, physics, and bioengineering programs by providing experience in use of bioinformatics software, recombinant DNA techniques, protein biochemistry, and other important techniques; revising and updating undergraduate and graduate lecture courses through new lectures, bringing the PI's background in protein structure and function to the biology department; developing a novel semester-long section for freshmen with new teaching materials and hands-on experiences that helps encourage students to consider a career in science. Additional outreach and teaching activities planned include judging science fairs at (predominantly minority) local K-12 schools and guest seminars at the ACA summer school.