The liquid-liquid phase separation of human molecular chaperone Hsc70 with poly(ethylene glycol): building a phase diagram

The observation that many proteins can undergo liquid-liquid phase separation (LLPS) to form membraneless organelles (MLOs) has given rise to plenty of investigations in vivo and in vitro around the world. Molecular chaperones are particularly interesting not only because they play an important role in protein folding, but also because they can participate in MLOs as scaffolds and/or clients without loosing function. Such an observation reinforces the importance of these molecules in regulating the LLPS of specific proteins, which cause the formation of aberrant MLOs under pathological conditions. In this work, we studied the propensity of the human heat shock cognate 71 kDa protein (Hsc70) to undergo LLPS in the presence of a macromolecular crowding agent. We demonstrate that Hsc70 is capable of phase separate with poly(ethylene glycol) at a large range of chaperone as well as PEG concentrations. To do that, a phase diagram was constructed by using turbidimetric titrations at 600 nm in a 96 well-plate. This approach brings the advantage of detecting phase separation at smaller concentrations, and it was supported by optical microscopy images. We expect that this approach will allow us to compare the phase behavior of other proteins with PEG, as well as under different conditions such as ionic strength, PEG molar mass, etc.