Fluorescence-Based Investigation of the Structure and Functional Dynamics of the Mitochondrial Protein Import Machinery

基于荧光的线粒体蛋白质输入机制的结构和功能动力学研究

• 批准号: 1024908
• 负责人: Nathan Alder
• 金额: $ 78.89万
• 依托单位: University of Connecticut
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1024908&HistoricalAwards=false
• 关键词: Fluorescence; Based; Investigation; Structure; Functional

Intellectual MeritMitochondria are organelles that play central roles in fundamental cellular processes including energy metabolism and programmed cell death. There are roughly 1000 (yeast) to 1500 (man) proteins resident within mitochondria, nearly all of which are synthesized outside the organelle and then trafficked to the mitochondrion by molecular complexes in the mitochondrial membrane. Our knowledge of the operation of these multi-component transport complexes has grown immensely in recent years. However, because these complexes are bound within the mitochondrial membranes, they are recalcitrant to many traditional techniques that are used to study protein structure and function. Hence, fundamental questions regarding the activity of these essential complexes have remained unaddressed. This project will employ a novel fluorescence-based approach to study the function and biogenesis of the central mitochondrial protein transporter, the TIM23 complex. Variants of the core channel-forming subunit (Tim23) labeled with small fluorescent probes at specific sites will be analyzed both in the context of native membranes (mitochondria isolated from yeast) and in reconstituted model membrane systems. Insights into the structural dynamics of the complex will be obtained using both environment-sensitive fluorescent probes and fluorescence energy transfer. Moreover, because labeled complexes are functionally active, this approach allows the analysis of structural changes as they occur in real time in response to different effectors. This innovative approach will not only enhance our understanding of how proteins are transported across biological membranes but, more generally, it will provide insights into the dynamics and interactions of membrane proteins and pave the way for future fluorescence-based investigations of other membrane protein complexes. Broader Impacts This research program will provide the basis for multiple educational and outreach components. Research training will include two teams of graduate and undergraduate students, each focusing on specific objectives. Collaborative interactions with cell biologists, molecular geneticists and biophysicists will foster a multidisciplinary training program. Educational activities of the project will include incorporation of research findings into two upper-division courses taught by the PI: "The Structure and Function of Biological Membranes" and "Principles of Cellular Bioenergetics". In addition, research will be integrated into a series of laboratory-based modular summer courses on the principles of fluorescence spectroscopy, giving undergraduates exposure to cutting-edge instrumentation and research on complex molecular systems. The scientific outreach component will include sponsorship of students from underrepresented groups through a Summer Research Program for Minority Undergraduates. In this program, highly motivated undergraduates from regional colleges attend a ten-week summer research course in which they are trained in multiple techniques and given the opportunity to present their findings at regional scientific meetings.

线粒体是细胞器，在基本的细胞过程中发挥核心作用，包括能量代谢和程序性细胞死亡。大约有1000（酵母）到1500（人）蛋白质存在于线粒体内，几乎所有的蛋白质都是在细胞器外合成的，然后通过线粒体膜上的分子复合物运输到线粒体。近年来，我们对这些多组分运输复合体的运作的了解大大增加。然而，由于这些复合物结合在线粒体膜内，因此它们与许多用于研究蛋白质结构和功能的传统技术不相容。因此，关于这些基本复合物的活动的基本问题仍然没有得到解决。该项目将采用一种新的基于荧光的方法来研究中央线粒体蛋白转运蛋白TIM23复合体的功能和生物起源。在特定位点用小荧光探针标记的核心通道形成亚基（Tim23）的变体将在天然膜（从酵母中分离的线粒体）和重构模型膜系统中进行分析。洞察复杂的结构动力学将获得使用环境敏感的荧光探针和荧光能量转移。此外，由于标记的复合物是功能活性的，这种方法允许分析结构变化，因为它们响应于不同的效应物在真实的时间内发生。这种创新的方法不仅将增强我们对蛋白质如何跨生物膜运输的理解，而且更普遍地说，它将提供对膜蛋白的动力学和相互作用的见解，并为未来基于荧光的其他膜蛋白复合物的研究铺平道路。更广泛的影响这个研究计划将提供多个教育和推广组成部分的基础。研究培训将包括两个研究生和本科生团队，每个团队都专注于特定目标。与细胞生物学家，分子遗传学家和生物药理学家的合作互动将促进多学科的培训计划。该项目的教育活动将包括将研究成果纳入PI教授的两门高年级课程：“生物膜的结构和功能”和“细胞生物能量学原理”。此外，研究将被整合到一系列基于实验室的模块化暑期课程的荧光光谱学的原则，让本科生接触到尖端的仪器和复杂的分子系统的研究。科学推广部分将包括通过少数民族大学生暑期研究计划赞助代表性不足的群体的学生。在这个项目中，来自地区大学的积极性很高的本科生参加为期十周的夏季研究课程，他们在其中接受多种技术的培训，并有机会在地区科学会议上展示他们的发现。