Project Details
Projekt
Conformational selection in liquid-liquid phase separation: Protein folding and aggregation pathways in the cytoplasm and stress granules
Applicant
Professor Dr. Simon Ebbinghaus
Subject Area
Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
Biophysics
Biophysics
Term
since 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 419071406
It is now clear that biomolecular phase separation plays a profound role in folding, aggregation and amyloid-associated pathologies. Under cell stress, condensates can sequester disease-related proteins leading possibly to a removal of toxic species from the cytoplasm ‘storing’ and ‘buffering’ them in condensates for degradation and shielding them from further aggregation. Such a mechanism would support protein quality control and thereby contribute to ensure protein homeostasis. On the other hand, it appears that once the ‘buffering’ capacity is overloaded, e.g. under prolonged stresses, the sequestration could be harmful to the condensate integrity leading to aberrant pathological phase transitions. The current knowledge of the link between condensation and aggregation is mainly derived from enrichment studies of disease-related proteins in condensates paired with the onset of aberrant phase transitions. The aim of our research in SPP2191 is to advance this knowledge by a consideration of the protein’s conformational states and dynamics along its folding and aggregation pathway. We share the aim to include conformational transitions (not limited to proteins) in analyzing phase separation processes with various research groups in SPP2191, leading to efficient research collaborations to establish common methods, model systems and theoretical models. The proposed research project is built on our preliminary work in the first funding period, in which we studied the sequestration of destabilized, but monomeric and reversibly-folded, superoxide dismutase 1 (SOD1) mutants by stress granules (SGs) (published results: N. Samanta et al., S. Ebbinghaus, Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability, J. Am. Chem. Soc., 2021.). We found that the partitioning between SGs and the cytoplasm is balanced by different factors such as protein folding stability, hydrophobicity and environmental conditions. The results sparked our proposed research objective to study how (mis-)folding and aggregation of disease-related SOD1 mutants further progresses in the mutual phases. To conduct this research, we propose to use specialized in-cell folding techniques such as confocal Fast Relaxation Imaging to quantitatively measure the thermodynamics and kinetics of the distinct pathway steps. We will be able to analyze folding and refolding, conversion to intermediate and misfolded states, as well as early self-association and aggregation of SOD1 in SGs in direct comparison to the mutual cytoplasmic processes in a single living cell. The project will advance the understanding of functional and dysfunctional condensates by revealing how SGs, in conjunction with other factors like molecular chaperones, could reshape folding and aggregation pathways to assist protein homeostasis, or conversely, how the integrity of SGs could be endangered by sequestration of different amyloidogenic species.
DFG Programme
Priority Programmes
