Development of metal ion-induced artificial protein liquid condensates with behavior tunability

Abstract

Phase separation of biomolecules into liquid droplet-like condensates is a key mechanism to form membraneless organelles, which spatiotemporally organize diverse biochemical processes in cells. To investigate the working principle of these biomolecular condensates, precise control of condensate properties is essential. Here, I design a strategy for metal ion-induced clustering of minimal protein modules to produce liquid protein condensates, the properties of which can be widely varied by simple manipulation of the protein clustering systems. A wide range of protein condensate properties such as droplet forming tendency, droplet morphology and density can be varied by adjusting the nature of receptor/ligand pairs, metal ions, metal/protein ratios, incubation time, and even spacing between interaction motifs. Utilizing developed protein condensate system, enhanced biochemical reaction rates inside condensates were observed. Also, the change of reaction rates inside condensates upon varying condensate viscosity tuned by scaffold inter-motif linker variation was examined. The present phase separation strategy provides highly versatile protein condensates, which will greatly facilitate investigation of molecular and structural codes of droplet-forming protePhase separation of biomolecules into liquid droplet-like condensates is a key mechanism to form membraneless organelles, which spatiotemporally organize diverse biochemical processes in cells. To investigate the working principle of these biomolecular condensates, precise control of condensate properties is essential. Here, I design a strategy for metal ion-induced clustering of minimal protein modules to produce liquid protein condensates, the properties of which can be widely varied by simple manipulation of the protein clustering systems. A wide range of protein condensate properties such as droplet forming tendency, droplet morphology and density can be varied by adjusting the nature of receptor/ligand pairs, metal ions, metal/protein ratios, incubation time, and even spacing between interaction motifs. Utilizing developed protein condensate system, enhanced biochemical reaction rates inside condensates were observed. Also, the change of reaction rates inside condensates upon varying condensate viscosity tuned by scaffold inter-motif linker variation was examined. The present phase separation strategy provides highly versatile protein condensates, which will greatly facilitate investigation of molecular and structural codes of droplet-forming proteins and the monitoring of biomolecular behaviors inside diverse protein condensates.ins and the monitoring of biomolecular behaviors inside diverse protein condensates.