CBE Doctoral Dissertation Defense: “Engineering Interactions Among Condensed Phases of Intrinsically Disordered Proteins” (Zhihui Su)

Abstract:

Proteins containing intrinsically disordered regions (IDRs) can undergo multivalent interactions that promote phase separation to form mesoscale condensates that function as membraneless organelles. As subcellular compartments, condensates hold promise as reaction vessels for biochemical applications in vitro, in protocells, and in living cells. However, precise valency control and multi-phase sub-compartmentalization are still inaccessible. This dissertation addresses the limitations of covalent assemblies, like fixed valency and surface fouling, and offers a new platform for noncovalent IDR multimerization and assembly as well as controlled chimeric or Janus multi-IDR assemblies. I used the LAF-1 RGG domain, the fused in sarcoma low complexity (FUS LC) domain, and the elastin-like polypeptide (ELP) domain as model IDR building blocks and assembled them with orthogonal heterodimeric parallel coiled-coil binding motifs to develop condensates with tunable IDR valency, size, critical temperature, composition, and topology. In this thesis, I first genetically fused parallel coiled-coils to the RGG domain on various termini to determine the structural organization for potent dimerization and condensate formation. I then established orthogonality across the entire NICP coiled-coil set to confirm specific and selective RGG domain dimerization. With these well-characterized coiled-coil interactions, I explored various coiled-coil placement strategies for higher-order IDR assemblies that allow modular design and have reduced fouling compared to covalently linked RGG domains with the same valency. Building on the multivalency, I studied interactions between RGG domains and FUS LC or ELP domains for multi-IDR assembly characterization and control. While RGG and FUS LC domains are naturally immiscible, I created a merged and chimeric dual-IDR phase with cognate coiled-coil interactions. Manipulating ELP’s LCST and RGG’s UCST with different ionic strength and salt concentrations, I defined precise control over interfacial organization, enabling the formation of Janus droplets, sub-compartmentalization, and merged chimeric phases. Taken together, this thesis elucidates design rules for engineering interactions among condensed phases of intrinsically disordered domains, presents a novel platform for multivalent and multi-component assembly with tunable spatial organization and condensate morphology, and sets the foundation for functional membraneless organelles in bioengineered cells and protocells.