Biophysical investigation of the liquid-liquid phase separation solvent interface.

Liquid-liquid phase separation (LLPS) is central to compartmentalisation of biochemical processes and allows co-localisation of a whole biological machine and its substrates at high local concentrations. This often dynamic and reversible assembly is formed by multivalent interactions between several biomolecules, and some instances involve low complexity sequences that have been linked to amyloid fibre formation. While the biophysical understanding of this phenomenon has recently been of high interest in the scientific community, the interactions of LLPS-forming proteins from the dilute phase with the interface to the condensed phase remain elusive. In this project we aim to dissect these transient interactions using state-of-the-art biophysical techniques. More specifically, we will use nuclear magnetic resonance (NMR), high-resolution-relaxometry (HRR) and an array of single-molecule fluorescence techniques to dissect up to atomic resolution and at multiple time-scales the transient interactions of the dilute phase proteins with the interface of the condensed state. We shall rely on the non-homologous end joining (NHEJ) system, that our laboratory has recently shown to exhibit LLPS in a broad range of conditions. Atomic-level dynamic information on the mechanisms for NHEJ phase separation and assembly could prove crucial both in the fundamental understanding of LLPS formation and growth, and in rational drug design aimed at preventing double-strand break repair by NHEJ in the frame of cancer treatment.