Studies on biological reactions inside protein droplets formed by liquid-liquid phase separation

Abstract

Recently, phase separation is known to play a role in the formation of various membraneless cellular organelles. These organelles are suggested as supramolecular assemblies of proteins and RNA molecules, that accumulate its comprising element and other biomolecules. This localization causes enrichment of the local concentration in phase-separated droplets. However, the detailed principle in increasing local concentration effects on biological kinetics is undiscovered. This study suggests behavior aspects of localized biomolecules in phase-separated droplets by a simplified model system. Using proline-rich motifs (PRM) and the SRC homology 3 (SH3) domain interaction, metal-induced protein phase separation scaffold was constructed as a model system. In the PRM-SH3-His (PSH) model scaffold, I develop interactable client proteins adjusted by the number of PRM. As a stable reaction product, isopeptide bond formation reaction was adopted as a target reaction. To follow reaction, fluorescent protein and dye were used. As a result, more interaction motifs cause higher local concentration. However, the overly high local concentration of scaffold protein and strong interaction like a nickel with 6x histidine tag are restricted molecular movement which comes up as decline of reactivity.