New fluorophores and labeling strategies for single molecule imaging

• Applicant: Dr. Maximilian Ulbrich
• Subject Area: Biophysics
• Term: from 2015 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 278267951

Interactions between proteins are the foundation of most biological processes inside cells. Many established biochemical methods aim at the determination of pairwise interactions but fail to capture complex assembly patterns that involve three or more components and their multiplicity in the formed complex. Membrane proteins are particularly difficult to analyze because of their low expression or their propensity to cluster when removed from the membrane environment.Previously, we had developed a single molecule method for counting subunits of a membrane protein complex and observation of direct interactions of the proteins in the membrane of a living cell. The method is based on imaging of bleaching steps from fluorescent proteins (FPs) that are genetically fused to the protein of interest, and on co-localization of FPs of two different emission wavelength. For more complex assembly patterns, a third or fourth wavelength is lacking, and when imaging membrane proteins that show fast diffusion in the cell membrane, the currently available red FPs are too dim.The aim of the proposed project is to extend the number of simultaneously useable fluorophores to three or four, and to establish new strategies that allow labeling of membrane proteins in live cells with organic dyes. For the FPs and organic dyes we will determine photophysical properties that are relevant for single molecule imaging like the fraction of non-functional fluorophores, the blinking behavior and the photostability under single molecule illumination conditions. Strategies for labeling with organic dyes will include the SNAP-tag, HALO-tag and nanobodies. Known multimeric membrane proteins and genetically fused FP concatemers will serve as scaffolds to immobilize the fluorophores in the environment of a living cell. The proposed work will not only help our laboratory to design our ongoing studies on membrane protein interactions more effectively, but provide an important resource for other researchers who use single molecule approaches involving fluorescent proteins for analyzing protein interactions.

• DFG Programme: Research Grants