Functional Dynamics and Energy Coupling Mechanisms of Mitochondrial Membrane Proteins

线粒体不仅在真核细胞内产生绝大多数能量，而且还调节细胞过程，如钙稳态、脂质合成和代谢物转运。由这个细胞器执行的重要功能直接取决于以质子电化学梯度的形式存储在其内膜上的势能。作为所有节能膜的特征，这种电势被解析为电场和跨脂双层的质子活性差异。本项目将解决线粒体内膜电化学电位的两个基本特征。首先，将研究由内膜的能量状态的改变驱动的膜蛋白结构的变化。新的，高分辨率的荧光为基础的方法将被用来阐明的方式，其中改变质子梯度和跨膜电场差异改变的构象的一个模型difera驻留蛋白。阐明这种机电耦合的基础对于理解节能膜中的蛋白质如何利用电化学势来执行细胞工作至关重要。第二，拓扑复杂的内膜的能量景观将被调查。使用精确靶向的pH传感和电致变色探针来测量局部离子梯度和电场，内膜能量分布的时间和空间异质性将以前所未有的分辨率测量。通过挑战当前跨膜区域的电化学电势平衡的教条，这项工作准备创建一个新的范式，用于理解过程的能量调节，如ATP的产生和双层结构的局部变化。这些进展，在目前的理解膜生物能量学将成为可能的技术创新，在这项研究中使用。更广泛的影响这个NSF赞助的工作将涉及两个研究团队的模型，旨在促进多学科的互动和发展的领导力在科学，强调学生的参与传统上代表性不足的STEM领域。这项工作还将有助于进一步开发和扩展模型膜系统构建和位点特异性多肽标记过程中的新研究工具，所有这些都将提供给科学界。该项目的教育推广部分是基于与康涅狄格大学早期大学体验计划合作的生物学暑期研究所系列。这一系列模块化的课堂和实验室课程为区域高中的教师提供专业发展，使他们了解研究和当前科学主题的进展，这些主题可以在随后的学年中融入自己的课程。随着这一方案的发展，该研究所将扩大到包括高中学生的暑期课程。总之，这一教育推广活动将与该项目的研究活动高度结合。此外，这一教学推广模式的成果将在教育出版物和会议讲习班中传播，作为有效帮助提高公众科学素养的一种手段。

INTELLECTUAL MERITMitochondria not only produce the vast majority of energy within eukaryotic cells, but also regulate cellular processes such as calcium homeostasis, lipid synthesis, and metabolite transport. The vital functions carried out by this organelle depend directly on the potential energy that is stored across its inner membrane in the form of a proton electrochemical gradient. Characteristic of all energy-conserving membranes, this potential is parsed as an electric field and a difference in proton activity across the lipid bilayer. This project will address two fundamental features of the mitochondrial inner membrane electrochemical potential. First, the changes in membrane protein structure that are driven by alterations in the energized state of the inner membrane will be investigated. Novel, high resolution fluorescence-based approaches will be employed to elucidate the manner in which alterations in the proton gradient and transmembrane electric field differentially change the conformation of a model mitochondria-resident protein. Elucidating the basis of such electromechanical coupling is vital to understanding how proteins in energy-conserving membranes harness the electrochemical potential to perform cellular work. Second, the energetic landscape of the topologically complex inner membrane will be investigated. Using precisely targeted pH-sensing and electrochromic probes to measure localized ion gradients and electric fields, the temporal and spatial heterogeneity of the inner membrane energetic profile will be measured with unprecedented resolution. By challenging the current dogma of electrochemical potential equilibrium across the membrane regions, this work is poised to create a new paradigm for understanding the energetic regulation of processes such as ATP production and local changes in the bilayer structure. These advances in the current understanding of membrane bioenergetics will be made possible by the technical innovations used in this research.BROADER IMPACTSThis NSF-sponsored work will involve two teams of researchers in a model designed to promote multidisciplinary interactions and development of leadership in the sciences, emphasizing the involvement of students traditionally underrepresented in the STEM fields. The work will also serve to further develop and expand novel research tools in the construction of model membrane systems and in the process of site-specific polypeptide labeling, all of which will be made available to the scientific community. The education outreach component of this project is based on the Biology Summer Institute series in cooperation with the University of Connecticut Early College Experience Program. This series of modular classroom and laboratory courses offers professional development to teachers from regional high schools, exposing them to advances in research and current scientific topics, which can be integrated into their own curricula during the subsequent academic year. As this program develops, the Institute will be expanded to include summer courses for high school students as well. Taken together, this education outreach will be highly integrated with the research activities of the project. Moreover, the outcomes of this pedagogical outreach model will be disseminated in educational publications and conference workshops as a means of effectively helping to raise scientific literacy of the public.