A multi-step nucleation process determines the kinetics of prion-like domain phase separation

Abstract

Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. Here, we combine rapid-mixing time-resolved small-angle X-ray scattering (SAXS) approaches to characterize the assembly kinetics of a prototypical prion-like domain with equilibrium techniques that characterize its phase boundaries and the size distribution of clusters prior to phase separation. We find two kinetic regimes on the micro- to millisecond timescale that are distinguished by the size distribution of clusters. At the nanoscale, small complexes are formed with low affinity. After initial unfavorable complex assembly, additional monomers are added with higher affinity. At the mesoscale, assembly resembles classical homogeneous nucleation. Careful multi-pronged characterization is required for the understanding of condensate assembly mechanisms and will promote understanding of how the kinetics of biological phase separation is encoded in biomolecules.

Liquid-Liquid Phase Separation in Nucleation Process of Biomineralization

“…Liquid-liquid phase separation (LLPS) is a popular interdisciplinary concept and is at the very core of the chemical (Freedman, 2020;Martin et al, 2020;Park et al, 2020;Riback et al, 2020;Iwata et al, 2021), biological (Ahn et al, 2021;Li RH et al, 2021;Deepankumar et al, 2021;Huang et al, 2021), and physical (Martin et al, 2021) processes of nature. Hundreds of high-level articles from many research fields focused on LLPS and tried to figure out its formation, performance, and regulation (Zhang et al, 2018;Alberti et al, 2019;Case et al, 2019;Gibson et al, 2019;Martin et al, 2020).…”

Competitive membrane wetting of polymer blends in artificial cells initiates phase separation and promotes fractionation

“…In addition, this multi-step nucleation-type phase separation corresponds the nucleation process of prion-like LLPS in the bulk. 30 Upon the membrane wetting, PEG6k and short dextran with a lower Mw appeared near the membrane covering the droplet surface, whereas the long dextran with a higher Mw localized at the droplet center, as illustrated in Figure 6b. 8,31 This implies that the compositional bias due to the membrane wetting can facilitate the phase separation of PEG/dextran blends near the critical point.…”

Melatonin: Regulation of Prion Protein Phase Separation in Cancer Multidrug Resistance

“…The regulation of prion functionality and conversion into toxic aggregates may be fundamentally propelled by LLPS [ 120 , 121 , 122 , 123 ], and the intrinsically disordered N-terminal region of the physiological PrP C has been shown to be necessary and sufficient for LLPS of PrP [ 266 , 267 ]. Large nucleation barriers enable deep supersaturation that favors the formation of toxic aggregates in Sup PrD while kinetic barriers for the formation of dynamic intracellular condensates are easily breached by PTMs and changes in salt, pH, and temperature during LLPS [ 251 , 268 , 269 ]. Nevertheless, fluctuations in RNA concentrations can modulate prion aggregation in a bimodal, concentration-dependent manner where high protein to RNA ratios stimulate aggregation and low ratios suppress condensate formation.…”

“…Phase separation is an evolutionarily conserved response used by living organisms to assemble biomolecular condensates as efficient adaptation to rapidly changing endogenous or exogenous stressors [ 190 , 196 ]. The formation of condensates during LLPS is a process of nucleation and growth constrained by an energy barrier that can usually be breached by thermodynamic nonequilibrium PTMs [ 269 , 302 ]. Many well-known targets of melatonin including NLRP3 inflammasome [ 303 , 304 , 305 ] and tumor suppressor protein p53 [ 306 , 307 , 308 ] contain prion-like IDRs that facilitate LLPS [ 265 , 309 , 310 , 311 ] and are regulated by ATP-dependent PTMs such as phosphorylation, ubiquitination, and SUMOylation [ 312 , 313 , 314 , 315 , 316 , 317 ], while DEAD-box RNA helicases such as DDX3X, which are tuned by RNA and ATP [ 318 ], can critically determine the outcome of prionoid LLPS in NLRP3 [ 310 ].…”