SGER: Proteomics-level Structural Biology of Transmembrane Proteins

SGER：跨膜蛋白的蛋白质组学水平结构生物学

• 批准号: 0432322
• 负责人: Constance Jeffery
• 金额: $ 9.27万
• 依托单位: University of Illinois at Chicago
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0432322&HistoricalAwards=false
• 关键词: SGER; Proteomics; level; Structural; Biology

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.

智力优势：许多重要的细胞活动，包括离子转运、细胞间通讯、囊泡转运、细胞结构的维持和宿主-病原体相互作用，都涉及嵌入细胞膜的蛋白质。 跨膜（TM）蛋白占已知蛋白的25%以上，并且是当今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，但对其结构和分子机制的了解远不如可溶性蛋白。疏水序列的存在使得难以表达和分离大量的这些蛋白质，并且使得它们难以用许多生物化学和结构方法来处理。这个探索性研究（SGER）项目的小额赠款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，而不是其他？更具体地说，是否有一个一般的物理障碍，以高水平的TM蛋白质的表达，或问题是由于更多的个别蛋白质的调节？改变生长条件能提高TM蛋白表达和膜定位吗？ 一个系统的蛋白质组学方法被用来测试的表达和膜定位的30个跨膜蛋白具有各种特性和各种生长条件下。 研究目标包括：（A）从模型细菌系统中选择30种靶蛋白，所述靶蛋白在预测的跨膜螺旋的数目、功能和其它特征方面不同，和（B）构建编码每种靶蛋白的表达载体以确定哪些蛋白质特征影响蛋白质表达和哪些蛋白质特征影响蛋白质在膜上的定位。该SGER项目正在使用30种蛋白质进行测试，以验证概念，然后根据结果，将使用更多蛋白质进行标准提案。 该项目的成功完成具有潜在的高影响力，因为它解决了获得足够量TM蛋白用于结构和分子机制研究的改进方法的需求。 虽然这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个基因组也为未来更大规模的系统研究奠定了基础，以开发TM蛋白的改进表达方法，以及这类重要蛋白的溶解，纯化和表征。这个SGER项目的更广泛的影响包括在实验室中培训和指导学生，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，培训和指导来自生物学，物理学和生物工程课程的各种本科生和研究生;通过新的讲座修订和更新本科生和研究生讲座课程，将PI的蛋白质结构和功能背景带到生物系;为新生开发一个新的学期部分，提供新的教材和实践经验，有助于鼓励学生考虑从事科学职业。计划开展的其他外联和教学活动包括在当地K-12学校（主要是少数民族学校）举办科学博览会，以及在ACA暑期学校举办客座研讨会。