Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity

Abstract

Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

“…[1,2] They play acrucial role for example in the compaction of polynucleotides and regulation of genetic expression. [7,22,23] Fori nstance,t heir formation and dissolution has been achieved by changes in pH, [5] temperature, [24][25][26] ionic strength, [27,28] and more recently by enzymemediated catalytic activity. [5][6][7] These membrane-free molecularly crowded compartments seques-ter functional biomolecules [5,8] and support enzymatic activity, [9,10] protein folding, [11] RNAc atalysis, [12,13] and cell-free protein expression.…”

“…[14] Polynucleotides have been used as scaffold components to assemble coacervate droplets, [15][16][17][18][19] and selective sequestration of guest polynucleotides has been demonstrated in preformed coacervates. [7,22,23] Fori nstance,t heir formation and dissolution has been achieved by changes in pH, [5] temperature, [24][25][26] ionic strength, [27,28] and more recently by enzymemediated catalytic activity. [7,22,23] Fori nstance,t heir formation and dissolution has been achieved by changes in pH, [5] temperature, [24][25][26] ionic strength, [27,28] and more recently by enzymemediated catalytic activity.…”

Physical Chemistry of Cellular Liquid‐Phase Separation

“…The insights and predictions of these experiments can then be tested in cells. For example, recent work using theory and experiment has shed light on the rules that govern various features of LLPS including spatial patterning of droplets . Other recent work has used mutagenesis to reveal the roles of side chain interactions underlying phase separation of the family of proteins that includes ALS‐linked protein Fused in Sarcoma (FUS) .…”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…[108] Aw ide class of naturallyo ccurring polymers capable of thermally driven simple coacervationi nw ater is that of elastomeric proteins such as elastinso rr esilins. [111] In ar ecent study,t hermally driven simplec oacervation of polynucleotides, based on purine-containings sDNA chains that weref ound to exhibit LCST-type behaviour,w as demonstrated. [111] In ar ecent study,t hermally driven simplec oacervation of polynucleotides, based on purine-containings sDNA chains that weref ound to exhibit LCST-type behaviour,w as demonstrated.…”

“…[160] Reproducings uch am ultilayered organisationi na ll-aqueous synthetic systems is very challenging, due to the low interfacial energies involved. [163] In anothere xample, spatially organised droplets with subcompartments were reportedi nR NA-protein systems via the rational design of RNA sequences and modulation of the interactions involved between components. [161] Dynamic vacuolisation was also observed in RNA/peptide coacervate droplets produced through DNA transcription, and attributed to ar eentrantp hase-transition process.…”