Impact of Macromolecular Crowding and Compression on Protein–Protein Interactions and Liquid–Liquid Phase Separation Phenomena

We determined the intermolecular interaction potential, V(r), of dense lysozyme solutions, which governs the spatial distribution of the protein molecules and the location of its liquid–liquid phase separation (LLPS) region, in various crowding environments applying small-angle X-ray scattering in combination with liquid-state theory. We explored the effect of polyethylene glycol (PEG) on V(r) and the protein's phase behavior over a wide range of temperatures and pressures, crossing from the dilute to the semidilute polymer regime, thereby mimicking all crowding scenarios encountered in the heterogeneous biological cell. V(r) and hence the protein–protein distances and the phase boundary of the LLPS region strongly depend on the polymer-to-protein size ratio and the polymer concentration. The strongest effect is observed for small-sized PEG molecules, leading to a marked decrease of the mean intermolecular spacing of the protein molecules with increasing crowder concentration. The effect levels off at intermolecular distances where the proteins' second hydration shells start to penetrate each other. Strong repulsive forces like hydration-shell repulsion and/or soft enthalpic protein-PEG interactions must be operative at short distances which stabilize the protein against depletion-induced aggregation, also at pressures as high as encountered in the deep sea, where pressures up to the kbar-level are encountered.