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.
Highlights
Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time
We have previously characterized the equilibrium phase behavior of A1-LCD as a function of temperature and have shown that it is well approximated by classic homopolymer mean-field models[31]
Similar to other prion-like domains[32], A1-LCD phase separation is enhanced with the addition of monovalent salts[33] and NaCl can be used as a parameter to control quench depth
Summary
Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. In bacteria[1,2] and eukaryotes[3], the selective condensation of subsets of biomolecules through liquid–liquid phase separation (LLPS) provides a means of spatial and temporal organization[4,5,6,7,8,9]. These condensates can rapidly form and dissolve in response to gradients in biomolecule concentration[7] or environmental stimuli[10,11]. At a given quench depth, the difference between the actual solution concentration and csat is referred to as the degree of supersaturation (σ)
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