Chapter 12: Integrative Methods to Investigate Chaperones in Regulating Protein Phase Separation and Aggregation

Protein liquid–liquid phase separation (LLPS) plays an essential role in driving the formation of membraneless organelles (MLOs) involved in different biological processes, dysregulation of which may lead to protein amyloid aggregation which is closely associated with different neurodegenerative diseases. Molecular chaperones are key players in maintaining the protein homeostasis of MLOs and regulating pathological protein aggregation. In the first half of this chapter, we summarized the emerging roles of different molecular chaperones in modulating protein LLPS as well as pathological aggregation. In the second half, we introduced different biophysical and biochemical methods for investigating how chaperones bind and modulate their client proteins in three different states including the diluted state, the dynamic phase separated state, and the aggregated fibrillar state. In the end, we pointed out the further direction in studying the chaperone–client interaction in cells by developing and integrating new methods.