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

线粒体是在包括能量代谢和细胞程序性死亡在内的基本细胞过程中起核心作用的细胞器。线粒体内大约有1000到1500种（酵母）蛋白质，几乎所有这些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物的操作的了解有了极大的增长。然而，由于这些复合物结合在线粒体膜内，它们对许多用于研究蛋白质结构和功能的传统技术具有抗性。因此，关于这些基本复合物的活性的基本问题仍然没有得到解决。该项目将采用一种新颖的基于荧光的方法来研究中心线粒体蛋白转运体TIM23复合物的功能和生物发生。用小荧光探针在特定位点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）和重建的模型膜系统中进行分析。利用环境敏感的荧光探针和荧光能量转移来深入了解该复合物的结构动力学。此外，由于标记的配合物在功能上是活跃的，这种方法允许分析结构变化，因为它们在响应不同的效应时实时发生。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，而且更一般地说，它将提供对膜蛋白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。更广泛的影响本研究项目将为多种教育和推广组成部分提供基础。研究训练将包括两组研究生和本科生，每组都侧重于特定的目标。与细胞生物学家，分子遗传学家和生物物理学家的协作互动将促进多学科培训计划。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荧光光谱原理模块化暑期课程中，让本科生接触到尖端仪器和复杂分子系统的研究。科学拓展部分将包括通过少数民族本科生暑期研究项目赞助来自代表性不足群体的学生。在这个项目中，来自地区高校的积极性很高的本科生参加了为期十周的暑期研究课程，在此期间，他们接受了多种技术方面的培训，并有机会在地区科学会议上展示他们的发现。

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.