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.

智力优点：许多重要的细胞活性，包括离子传输，细胞 - 细胞通信，囊泡传输，细胞结构的维持和宿主 - 死亡 - 病原体相互作用，都涉及嵌入细胞膜中的蛋白质。 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蛋白的折叠和/或活性不当会在正常细胞过程中引起严重的故障。 尽管TM蛋白具有巨大的重要性，但对它们的结构和分子机制的了解远不如可溶性蛋白。疏水序列的存在可能使得很难表达和分离大量这些蛋白质，并使其对许多生化和结构方法难治性。这项用于探索性研究（SGER）项目的小赠款解决了一个总体问题：为什么重组蛋白表达方法会导致某些TM蛋白的过表达和纠正膜定位，而不是其他TM蛋白？更具体地说，是否存在高水平TM蛋白表达的一般物理障碍，还是由于调节单个蛋白质而导致的问题？改变生长条件是否可以改善TM蛋白表达和膜定位？ 一种系统的蛋白质组学方法用于测试30种具有多种特征并在各种生长条件下的跨膜蛋白的表达和膜定位。 研究目的包括：（a）从模型细菌系统中选择30种靶蛋白，该系统在预测的跨膜螺旋，功能和其他特征的数量上有所不同，以及（b）构建编码每个靶蛋白的表达向量，以确定哪种蛋白质特征会影响蛋白质表达以及哪种蛋白质特征影响蛋白质的蛋白质特征对膜的局部局限。该SGER项目正在使用30种蛋白质进行测试，以测试概念证明，然后，根据结果，标准建议将随后使用大量的蛋白质。 该项目的成功完成可能具有很高的影响，因为它解决了需要改进的方法，以获取足够数量的TM蛋白来研究结构和分子机制。 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是一所城市大学，其学生化妆反映了社区，其中包括许多来自具有适度收入的家庭的学生，许多学生是家人上大学的第一个学生，以及来自代表性不足的团体的学生。 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经验，培训和指导来自生物学，物理和生物工程计划的各种本科生和研究生；通过新的讲座修改和更新本科和研究生讲座课程，将PI的蛋白质结构和功能背景带给生物学系；通过新的教学材料和动手实践经验为新生开发一个新颖的学期部分，有助于鼓励学生考虑科学职业。计划的其他推广和教学活动包括在ACA暑期学校（主要是少数）本地K-12学校（主要是少数）的科学博览会和来宾研讨会。