Conformational Selection in Liquid-Liquid Phase Separation: Integrating Protein Folding, Misfolding and Stress Granule Formation

Intracellular membraneless organelles (MOs) form dynamically and thermodynamically reversible via liquid-liquid phase separation (LLPS). The molecular processes that drive MO formation are largely unknown, but recent deletion experiments showed that their formation and function is often associated with intrinsically disordered protein domains. Their flexibility and ability for multiple weak protein-protein interactions (PPIs) seem to be crucial to form a protein-rich separated phase. Stress granules (SGs) are MOs that are formed in response to heat, cold, or oxidative cell stresses. They contain pathogenic forms of proteins that are associated with neurodegenerative diseases su as the enzyme superoxide dismutase (SOD1) involved in familiar amyotrophic lateral sclerosis. Pathogenic forms of SOD1 are destabilized compared to the wildtype by point mutations, show an increased propensity to aggregate, and can transform SGs into an aberrant solid-like state. In the proposed research, we want to understand how the processes of SOD1 unfolding, misfolding and aggregation are coupled to SG formation in health and disease conditions by a complementary in vitro, in vivo and in silico study. We focus on the early unfolding events of SOD1 and propose that the unfolded state strengthens the PPI network to control SG formation. The results could provide a fundamentally new understanding of the function of SGs in stress and diseas conditions associated with protein misfolding. Our research includes the development of a novel method to quantify how conformational changes of biomolecules contribute to control LLPS.

Current State of Research

Recently, we constructed a SOD1 folding reporter, including 10 disease-related mutants, and studied the respective in-cell folding stability. Based on this work, we will establish a thermodynamic and a kinetic assay to simultaneously measure SOD1 folding and SG formation in single cells. These assays will provide quantitative read-outs of how unfolded and misfolded states of SOD1 participate in PPIs controlling SG formation. To interpret the kinetic and thermodynamic data on the molecular level, we will reconstitute the system in vitro, studying varying concentrations of cosolutes. Finally, we study cellular SOD1 folding and SG regulation by biological factors as well as physicochemical factors (e.g. chaperons or osmotic pressure). We are excited to collaborate with experts in SPP 2191 to unravel the molecular details and functional aspect of LLPS. We further contribute a novel functional assay that can be used to study a broad range of molecular conformational switches that control LLPS in cells and multicellular organisms.