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

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.