Complex Coacervation of Disordered Proteins: focus on α-Synuclein and Polyamines
by Matthew Percival
Date of Examination:2024-10-30
Date of issue:2025-10-28
Advisor:Prof. Dr. Markus Zweckstetter
Referee:Prof. Dr. Markus Zweckstetter
Referee:Dr. Loren Andreas
Referee:Dr. Johannes Soeding
Persistent Address:
http://dx.doi.org/10.53846/goediss-11583
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Abstract
English
The discovery of phase-separated bodies in cells has revealed an additional mesoscale layer of organisation in cellular and molecular biology. The enrichment of RNA in these biomolecular condensates suggests that charge plays a key role in phase separation. Complex coacervation—a type of polymer phase separation—occurs in solutions of oppositely charged polyelectrolytes. In this project, we investigated the role of naturally occurring, cellularly abundant polyamines as cationic binding partners, given their close association with RNA biology and their ability to induce complex coacervation with nucleic acids. Unlike RNA, many proteins have non-uniform charge distributions and complex primary sequences, where the effects of charge must be considered alongside other factors, such as hydrophobicity and charge patterning. Whether these factors can similarly drive complex coacervation of intrinsically disordered proteins (IDPs) remains unclear. To explore this, we studied α-synuclein (αSyn) and G3BP1. G3BP1 is a stress granule scaffold protein whose condensation is driven by a combination of homotypic and heterotypic interactions. Its long acidic intrinsically disordered region (IDR1) suggests that interactions with cationic species may be as critical as its RNA-binding motif. Similarly, the role of αSyn’s acidic C-terminus in modulating phase separation and its connection to amyloid aggregation remain poorly understood. We find that PolyU RNA and spermine form stoichiometry-dependent complex coacervates that are highly sensitive to temperature and ionic strength, consistent with a classical complex coacervation process. Spermine also promotes phase separation of αSyn through charge interactions with its acidic C-terminus. This process is strongly dependent on αSyn- to-spermine stoichiometry, increases as the isoelectric point of αSyn is approached, and decreases as polyamines lose charge. Phase separation is abolished at NaCl concentrations above 200 mM, highlighting the electrostatically driven nature of the process. Importantly, heterotypic phase separation accelerates αSyn aggregation. Polyamines with weaker net charge relative to spermine interact more weakly with αSyn, resulting in reduced phase separation. IV Our results identify the C-terminus of αSyn as the scaffold domain for phase separation and show that multivalent polyamines can promote condensation at physiological concentrations. In sub-saturated solutions, clusters are preferentially formed, which persist within condensates, indicating electrostatic nanocluster formation and microstructural organisation. RNA, which binds spermine more strongly than αSyn, can sequester spermine from the protein, exposing the C-terminus and promoting miscibility. Finally, we show that G3BP1 interacts with spermine via its acidic IDR1, lowering the protein concentration required for phase separation. These findings provide mechanistic insight into how electrostatic interactions and multivalent cations regulate the phase separation of IDPs and stress granule scaffold proteins in cells.
Keywords: αSyn; Polyamines; Phase separation
