Tuning phase transitions of aqueous protein solutions by multivalent cations

In the presence of trivalent cations, negatively charged globular proteins show a rich phase behaviour including reentrant condensation, crystallisation, clustering and lower critical solution temperature metastable liquid–liquid phase separation (LCST–LLPS). Here, we present a systematic study on how different multivalent cations can be employed to tune the interactions and the associated phase behaviour of proteins. We focus our investigations on the protein bovine serum albumin (BSA) in the presence of HoCl3, LaCl3 and YCl3. Using UV-Vis spectroscopy and small-angle X-ray scattering (SAXS), we find that the interprotein attraction induced by Ho3+ is very strong, while the one induced by La3+ is comparatively weak when comparing the data to BSA–Y3+ systems based on our previous work. Using zeta potential and isothermal titration calorimetry (ITC) measurements, we establish different binding affinities of cations to BSA with Ho3+ having the highest one. We propose that a combination of different cation features such as radius, polarisability and in particular hydration effects determine the protein–protein interaction induced by these cations. Our findings imply that subtle differences in cation properties can be a sensitive tool to fine-tune protein–protein interactions and phase behaviour in solution.