Super Time-resolved Fluorescence Anisotropy with Switchable States

ended

Viable experimental techniques able to reveal and quantify protein-protein interactions and protein conformational changes can have a significant impact on cell biology and drug discovery. Fluorescence anisotropy (FA) has been widely employed in biomedical research as a tool for high-throughput screening applications, to study the binding of small molecules to protein and characterize protein-protein interaction. Despite the enormous potential of the FA technique, the major limiting factor is the inability of probing the system past the fluorescence lifetime, which in the most favorable cases lasts for a few nanoseconds, setting an upper limit to the time scales that can be addressed with the technique, which translates in an upper limit of few nanometers of molecular size. Reversibly switchable fluorescent transitions have the potential to revolutionize the capability of FA tools for the study of large molecular aggregates, overcoming the limits imposed by the finite fluorescence lifetime, and providing a practical and highly sensible way of measuring rotational diffusion processes with a practically unlimited upper bound on molecular sizes. This proposal aims to develop a novel fluorescent anisotropy technique, named Super Time-resolved Anisotropy with Reversibly Switchable States (STARSS), designed to measure rotational mobility all-across the time scale from nano- to micro-seconds, which will enable to discern clusters from free rotating molecules in situ and with high angular precision. The coupling of STARSS observables and microscopy will provide a powerful tool to reveal the dynamics of protein complexes inside the compartments of living cells, shedding new light on a multitude of biological processes.