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

• Published in: Nature communications 2021-07, Vol.12 (1), p.4513-4513, Article 4513
• Main Authors: Martin, Erik W., Harmon, Tyler S., Hopkins, Jesse B., Chakravarthy, Srinivas, Incicco, J. Jeremías, Schuck, Peter, Soranno, Andrea, Mittag, Tanja
• Format: Article
• Language: English
• Subjects: 631/45/535/1261; 631/57/2269; 631/57/2272/1590; 631/57/2272/2276; Affinity; Algorithms; Assembly; BASIC BIOLOGICAL SCIENCES; Biomolecules; Clusters; Complexity; Domains; Humanities and Social Sciences; Intrinsically disordered proteins; Kinetics; Liquid phases; Microscopy, Confocal; Models, Chemical; Monomers; multidisciplinary; Nucleation; Phase separation; Prions - chemistry; Protein Conformation; Proteins; SAXS; Scattering, Small Angle; Science; Science (multidisciplinary); Size distribution; Small angle X ray scattering; Supramolecular assembly; Thermodynamics; Time; X-Ray Diffraction - methods; X-ray scattering
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-24727-z

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. The nucleation mechanisms of biological protein phase separation are poorly understood. Here, the authors perform time-resolved SAXS experiments with the low-complexity domain (LCD) of hnRNPA1 and uncover multiple kinetic regimes on the micro- to millisecond timescale. Initially, individual proteins collapse. Nucleation then occurs via two steps distinguished by their protein cluster size distributions.