Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences

• Published in: Biochemistry (Easton) Vol. 61; no. 22
• Main Authors: Garabedian, Mikael V., Su, Zhihui, Dabdoub, Jorge, Tong, Michelle, Deiters, Alexander, Hammer, Daniel A., Good, Matthew C.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society (ACS) 02.08.2022
• Subjects: BASIC BIOLOGICAL SCIENCES; Condensation; Dimerization; Liquids; Monomers; Peptides and proteins
• ISSN: 0006-2960; 1520-4995

Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid–liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Furthermore, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over phase separation of RGG domains fused to FRB and FKBP in the presence of dimerizer. Further, using a photocaged dimerizer, we achieve optically induced condensation both in cell-sized emulsions and within live cells. Collectively, these modular tools allow multiple strategies to promote phase separation of a common core IDR for tunable control of condensate assembly.