Periodic Reporting for period 1 - STARSS (Super Time-resolved Fluorescence Anisotropy with Switchable States)
Okres sprawozdawczy: 2020-04-01 do 2022-03-31
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. However, with state-of-the-art techniques, it is very challenging to precisely, efficiently, and specifically follow these processes directly in a physiologically relevant system. An effective tool is the use of fluorescence anisotropy techniques that studies rotational diffusion properties of biological systems, providing direct insight into biological processes such as molecular binding, enzymatic activity, and protein assembly.
The novel approach developed during the project is called selective time-resolved fluorescence anisotropy with reversible with switchable states (STARSS), and it aims to measure protein complex formation in living cells overcoming the limits imposed by the photo-physics of fluorescence. STARSS takes inspiration from super-resolution resolution techniques in fluorescence microscopy, and it translates these powerful concepts into improved angular resolution and temporal range in rotational studies. The main objectives of the project were to develop a prototype optical system able to perform the experiments, screen for the best labeling solution for the samples, and develop proof-of-concept applications to showcase the potential of the technique.
The novel approach developed during the project is called selective time-resolved fluorescence anisotropy with reversible with switchable states (STARSS), and it aims to measure protein complex formation in living cells overcoming the limits imposed by the photo-physics of fluorescence. STARSS takes inspiration from super-resolution resolution techniques in fluorescence microscopy, and it translates these powerful concepts into improved angular resolution and temporal range in rotational studies. The main objectives of the project were to develop a prototype optical system able to perform the experiments, screen for the best labeling solution for the samples, and develop proof-of-concept applications to showcase the potential of the technique.
Two different prototype optical implementations of the STARSS concept were developed: (i) a STARSS-RESOLFT optical system that was used to measure samples containing green reversibly switchable fluorescence proteins; and (ii) a STARSS-STED optical system that was used to measure samples labeled with dyes able to undergo efficient stimulation emission depletion using light in the red-NIR region of the electromagnetic spectrum. The synergy between optics and the use of smart fluorescent probes were the key ingredients for successfully developing the STARSS technique. The validation of the optical setup was thus coupled with the screening and characterization of the best-performing fluorescent probes. Several proof-of-concept applications were developed, such as (i) the study of chromatin compaction and aggregation in the tens to hundreds nanometer scale, (ii) measurement of the maturation state of virus-like HIV-1 particles without the necessity of imaging and resolving the inner structure of the particles, (iii) and the study of the aggregation state and properties of the activity-regulated cytoskeleton-associated protein in live-cells.
The STARSS technique demonstrated to be a working concept for overcoming a few key limitations in fluorescence anisotropy experiments, allowing to extend the possibility of rotational diffusion studies of protein complexes in physiologically relevant conditions. Overall, the method developed during the project has been critical for understanding the fundamental difference between freely tumbling proteins and their interaction in higher-level structures in several example applications. The STARSS concept was implemented in set-ups using focused light, exploiting all the benefits of working in a confocal design. However, future implementation can be designed with simplified optics and tailored for screening studies. This is possible because the STARSS read-out does not lose content when moving away from the single-molecule and fluctuation regime. STARSS is also complementary to Förster resonance energy transfer studies where super-complexes form at distances above the Förster radius (1-10 nm), or where it is not possible to achieve a correct ratio of expression between donor and acceptor probes. The new method has the potential to remarkably enriching the library of problems studied with fluorescence anisotropy readouts, while delivering new tools that could be directly engineered in high-throughput screening applications, with a substantial impact on drug discovery.