Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences

Abstract

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. Here, 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.

Assembling membraneless organelles fromde novodesigned proteins

“…Others have reported the successful engineering of natural condensing proteins in cells to explore the potential of MLOs in synthetic biology. 12,15,17,18,51,52 Recent examples have demonstrated the capability for condensates to augment cells with artificial functions approaching the complexity of natural organelles, 14,53 and to generate compartments that can be modulated through rational changes to their scaffold proteins. 16 Engineered condensates have also incorporated designed enzymatic reactions, creating functional MLOs.…”

Strategies for Conditional Regulation of Proteins

“…Molecular biology techniques are currently being used to construct fusion proteins of any POI with an IDD to allow controlled phase separation. Stimuli such as light and small molecules are being explored for conditional regulation. − Though LLPS is a powerful tool for protein regulation, it should be noted that this process can initiate protein aggregation and misfolding, thus leading to many problems. A successful strategy for conditional regulation must be reversible and maintain protein quality after LLPS.…”

Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility

“…Alternatively, monomers can also be potentially sequestered away into stable oligomers, which could hinder LLPS 23 . Oligomerization has also been harnessed to allow the formation of de novo condensates [24][25][26][27] . In one such system, the optogenetic Corelet technology, light-dependent oligomerization of IDRs and other proteins enabled the quantitative mapping of phase diagrams in living cells, and has been utilized to probe the biophysical properties of condensate nucleation and coarsening behavior 28,29 .…”