Disentangling liquid-liquid phase separation and aggregation of intrinsically disordered proteins

Disentangling liquid-liquid phase separation and aggregation of intrinsically disordered proteins Disentangling liquid-liquid phase separation and aggregation of intrinsically disordered proteins Proteins and nucleic acids can physically partition without membranes through a liquid-liquid phase separation (LLPS) process. The structural assembly behaves like highly-concentrated liquid droplet, therefore providing dynamic environment for biomolecular reactions requiring specific protein concentrations. Many of the LLPS-enabled proteins are intrinsically disordered proteins (IDPs), lack of a well-defined folded structure. In addition, some are known to also aggregate when varying solvent conditions or introducing mutations. These observations raise key questions: how the functional dynamic droplets transform into the pathological solid aggregates. However until recently, there is limited work focusing on the principles able to distinguish between droplets and aggregates, which is essential in designing novel strategies disrupting only pathological aggregates but functional liquid droplets. In this proposed work, we will focus on the fundamental structure (dis)similarities of the two assembly states of an RNA-binding protein FUS, the N-terminal low complexity domain of which has been shown to form both liquid droplets and solid aggregates. For designing computational models to investigate structure properties of LLPS and aggregation, FUS is the most appropriate and the only system with rich experimental data on both assembly states. We propose to investigate the structure properties of FUS in both its dispersed and condensed phases, with and without interacting with its interaction partners. Ultimately, we aim at quantitatively assessing the impact of droplet formation on its aggregation preference. Towards these, we will implement an innovative combination of technologies from biophysics and spectroscopy, including all-atom explicit solvent and coarse-grained simulations, together with NMR (Nuclear magnetic resonance), FRET (Frster resonance energy transfer) and DLS (dynamic light scattering) through collaborating with four leading experimental groups in studying IDPs. We expect such a unique set of technologies to reach an unprecedented level of structure details on FUS LLPS and aggregation.