Enzymatic Regulation of Protein–Protein Interactions in Artificial Cells

摘要: Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein–protein or protein–nucleic acid interactions, which are often regulated by post‐translational modifications. However, the mechanisms behind these dynamic, affinity‐based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14‐3‐3 scaffold protein to study enzymatically regulated recruitment of 14‐3‐3‐binding proteins is presented, which mostly bind in a phosphorylation‐dependent manner. Synthetic coacervates are efficiently loaded with 14‐3‐3, and phosphorylated binding partners, such as the c‐Raf pS233/pS259 peptide (c‐Raf), show 14‐3‐3‐dependent sequestration with up to 161‐fold increase in local concentration. The c‐Raf domain is fused to green fluorescent protein (GFP …