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）开发了一种先进的基于荧光的方法和附带的用户友好型计算机程序，用于快速准确地确定活细胞中膜蛋白的超分子组装或低聚物的大小、丰度、稳定性、几何形状和功能角色。由此产生的基础设施将对大量研究人员产生重大影响，这些研究人员对鉴定和可视化蛋白质-蛋白质相互作用的复杂网络感兴趣，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。精心设计的外展计划将让高中和小学的学生亲自动手学习科学。这项研究将大大扩大威斯康星大学高中生研究实习计划的高中研究实习生的项目范围，该计划已经有来自威斯康星州东南部两所高中的41名学生（主要是少数民族）参加了为期三个月的研究实习。此外，这项研究将通过“脚踏实地的实验物理”（DEEP）计划，大大扩大学习应用于生物研究的光和光谱相关概念的机会，该计划迄今已招收了165多名小学生。即将开发的方法，称为强度波动和共振能量转移（iFRET），将整合荧光标记蛋白质的发射强度波动的细胞内测量，以确定低聚物的大小，并测量低聚物内连接到原聚物的光激发和其他非激发荧光标记之间发生的能量转移，以确定原聚物之间的距离。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的精湛结合，以及研究各种膜受体和转运蛋白寡聚化的基于荧光的方法的开发。这一创新将通过开发新的方法和专用软件来显著改善现有的基础设施，用于与各种光学显微镜兼容的数据分析。在初始阶段，研究将依靠威斯康星大学密尔沃基分校生物物理微光谱学设施提供的光谱分辨双光子微光谱学，该设备由美国国家科学基金会主要研究仪器基金开发，使用在各种环境条件下活细胞中表达的典型膜受体来实现新方法。为了进一步实现这类研究的民主化和技术的广泛传播，新方法将在威斯康星大学密尔沃基分校的其他实验室和合作机构的通用双光子显微镜和共聚焦显微镜上实施。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.