IIBR Research Methods: Probing the structure, abundance, dynamics, and function of protein complexes within their cellular environment

威斯康星大学密尔沃基分校（UWM）获得了一项奖项，以开发一种先进的基于荧光的方法和附带的用户友好的计算机程序，用于快速准确地确定活细胞中膜蛋白的超分子组装体或低聚物的大小，丰度，稳定性，几何形状和功能作用。由此产生的基础设施将显著影响有兴趣识别和可视化蛋白质-蛋白质相互作用的复杂网络的大型研究人员社区，并了解寡聚化在重要生物过程中的作用，如细胞信号传导，细胞间通讯，心脏调节，宿主-病原体相互作用和植物根系发育。此外，这项研究将为研究生和本科生创造一个良好的环境，以实践跨学科的科学方法，并参与开发未来的技术。精心设计的推广计划将涉及高中和小学学生动手学习科学。这项研究将大大拓宽项目的范围提供给高中研究实习生就读于UWM研究实习计划高中生，其中已经涉及41名学生（主要是代表性不足的少数民族）从两个东南威斯康星州高中在三个月的研究实习在其存在的七年。此外，这项研究将大大扩大学习与光和光谱学相关的概念的机会，适用于生物调查，通过脚踏实地的实验物理（或深度）计划，该计划迄今已招收超过165名小学生。该方法被开发，称为强度波动和共振能量转移（iFRET），将整合荧光标记蛋白质发射强度波动的细胞内测量，以确定寡聚体尺寸，并测量连接到寡聚体内原聚体的光激发和其它未激发荧光标记之间发生的能量转移，以确定原聚体间距离。iFRET的实施将建立在强有力的初步结果和团队在荧光蛋白合成和光物理表征方面的专业知识以及开发用于研究各种膜受体和转运蛋白寡聚化的基于荧光的方法的精湛组合的基础上。这项创新将通过开发与各种光学显微镜兼容的新方法和专用数据分析软件来显着改善现有基础设施。在其初始阶段，该研究将依赖于UW-Milwaukee的生物物理显微光谱设施中可用的光谱分辨双光子显微光谱仪，该仪器由NSF主要研究仪器基金开发，以使用在各种环境条件下活细胞中表达的典型膜受体来实施新方法。为了使这类研究进一步民主化和更广泛地传播这项技术，这种新方法将在威斯康星州大学其他实验室的通用双光子显微镜和共焦显微镜上实现。密尔沃基和合作机构。该奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查的支持的搜索.

An award is made to the University of Wisconsin-Milwaukee (UWM) to develop an advanced fluorescence-based method and accompanying user-friendly computer programs for rapidly and accurately determining the size, abundance, stability, geometry, and functional roles of supramolecular assemblies, or oligomers, of membrane proteins in living cells. The resulting infrastructure will significantly impact the large community of researchers interested in identifying and visualizing complex networks of protein-protein interactions and understanding the role of oligomerization in important biological processes, such as cellular signaling, intercellular communication, heart regulation, host-pathogen interactions, and plant root development. In addition, this research will create an excellent environment for graduate and undergraduate students to practice an interdisciplinary approach to science and to become involved in developing the technology of the future. Elaborate outreach programs will involve high school and elementary school students in hands-on learning of science. The research will significantly broaden the range of projects available to the high-school research interns enrolled in the UWM Research Internship Program for High School Students, which has already involved 41 students (mostly under-represented minorities) from two southeast Wisconsin high schools in three-month-long research internships during its seven years of existence. Furthermore, this research will significantly expand the opportunities for learning concepts related to light and spectroscopy as applied to biological investigations, through the Down-to-Earth Experimental Physics (or DEEP) Program, which has enrolled to date over 165 elementary school students.The method to be developed, termed intensity Fluctuation and Resonance Energy Transfer (iFRET), will integrate in-cell measurements of fluctuations in the emission intensity of fluorescently labeled proteins, to determine oligomer sizes, with the measurement of energy transfer occurring between an optically excited and other unexcited fluorescent tags attached to protomers within an oligomer, to determine inter-protomeric distances. The implementation of iFRET will build on strong preliminary results and the team’s exquisite combination of expertise in the synthesis and photo-physical characterization of fluorescent proteins and the development of fluorescence-based methods for studying oligomerization of various membrane receptors and transporters. This innovation will significantly improve upon the existing infrastructure by developing new methodology and dedicated software for data analysis compatible with a variety of optical microscopes. In its initial stage, the research will rely on the spectrally resolved two-photon microspectroscopes available in the Biophysical Microspectroscopy Facility at UW-Milwaukee, developed with NSF Major Research Instrumentation funds, to implement the new method using a typical membrane receptor expressed in living cells under various environmental conditions. For further democratization of this kind of research and broader dissemination of the technology, the new method will then be implemented on general-purpose two-photon microscopes and confocal microscopes available in other labs at the University of Wisconsin-Milwaukee and partnering institutions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.