Comprehensive analysis of the structural changes behind the phase-separation of proteins

The event of liquid-liquid phase separation (LLPS) is earning more and more scientific interest in both physiological and pathological processes. According to earlier observations the phase separation most often occur through the fuzzy interactions that disordered protein
segments form with RNA molecules. So far few attempts were made to decipher the structural state of the proteins inside of the phase separated droplets and not all of these were successful. The main aim of our collaboration is to establish appropriate methods for the characterization of the structure and structural changes of the proteins inside of the droplets. For this, we plan to use two different systems. One is the PR repeat peptide that has been shown to undergo LLPS in vitro and the other is the ERD14 protein that is capable of droplet formation in the presence of RNA. We plan to use different methods to study the LLPS. These are DLS (Dynamic Light Scattering), SAXS (Small-angle X-ray Scattering), FTIR (infrared spectroscopy), FF-TEM (Freeze-fracture transmission electron microscopy) and fluorescence microscopy techniques. Using DLS, we will be able to define the size-distribution of the droplets under different conditions. SAXS and FTIR will give information about the structure of the proteins inside the droplets. Larger inner structures, layers within the droplets can be visualized using FF-TEM and different fluorescence microscopy measurements. Our results will be important additions to the understanding of the molecular processes behind the liquid-liquid phase separation.

Ágnes Tantos - Krisztián Szigeti - Zoltán Varga