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)被授予一项奖项，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，用于快速和准确地确定活细胞中膜蛋白的超分子组件或低聚物的大小、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大型研究人员社区，他们有兴趣识别和可视化蛋白质-蛋白质相互作用的复杂网络，并了解寡聚在重要生物过程中的作用，如细胞信号、细胞间通信、心脏调节、宿主-病原体相互作用和植物根发育。此外，这项研究将为研究生和本科生创造一个良好的环境，让他们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生和小学生亲自动手学习科学。这项研究将极大地扩大参加威斯康星州州立大学高中生研究实习计划的高中研究实习生的项目范围，在该计划存在的七年中，已经有来自威斯康星州东南部两所高中的41名学生(主要是代表不足的少数族裔)参加了为期三个月的研究实习。此外，这项研究将极大地扩大学习与光和光谱学相关概念的机会，将其应用于生物学研究，通过扎实的实验物理(或深)计划，迄今已招募超过165名小学生。将开发的方法，称为强度波动和共振能量转移(IFRET)，将整合细胞内测量的荧光标记的蛋白质发射强度的波动，以确定齐聚体大小，与测量光激发和其他未激发的荧光标记之间发生的低聚物之间的能量转移，以确定原粒之间的距离。IFRET的实施将建立在强大的初步结果和该团队在荧光蛋白的合成和光物理表征以及研究各种膜受体和转运体齐聚的基于荧光的方法方面的专业知识的精妙组合的基础上。这项创新将通过开发新的方法和与各种光学显微镜兼容的数据分析专用软件，大大改进现有的基础设施。在初始阶段，这项研究将依靠密尔沃基大学生物物理显微光谱学设施中可用的光谱分辨双光子显微光谱仪，该设施由NSF重大研究仪器基金开发，利用在各种环境条件下在活细胞中表达的典型膜受体来实施新方法。为了进一步实现这类研究的民主化和技术的更广泛传播，新方法将在威斯康星大学密尔沃基分校和合作机构的其他实验室提供的通用双光子显微镜和共聚焦显微镜上实施。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.