Tuning phase transitions of aqueous protein solutions by multivalent cations

Abstract

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 HoCl 3 , LaCl 3 and YCl 3 . Using UV-Vis spectroscopy and small-angle X-ray scattering (SAXS), we find that the interprotein attraction induced by Ho 3+ is very strong, while the one induced by La 3+ is comparatively weak when comparing the data to BSA-Y 3+ 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 Ho 3+ having the highest one. We propose that a combination of different cation features such as radius, polarisability and in particular hydration effects determine the proteinprotein 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.

Multivalent ions and biomolecules: Attempting a comprehensive perspective

“…The width of the reentrant regimes, the lower critical transition temperature as well as the overall strength of the interaction can be strongly influenced by the type of multivalent cation. [59] Pronounced anion as well as solvent isotope effects on the phase behaviour of protein-multivalent salt systems have also been shown. [96,296] Generally, metastable LLPS in protein solutions is of specific interest due to its connection to protein crystallisation.…”

“…[54] While ion-ion correlations might add a finite contribution to the protein-ion interaction, other more specific effects appear to be more relevant. [58] An interesting point concerns competing-ion and co-ion effects, both for different multivalent ions as well as for a given species of multivalent ions [59] in the presence of a monovalent ion. [60] We shall mention that ion effects and in particular multivalent effects are also connected with the pH of the system, but unless explicitly stated otherwise, the effects we are discussing are dominated by the charge itself, and the pH is a secondary (although quantitatively important) effect.…”

“…Non-trivial trends in the protein binding behaviour of lanthanide and yttrium cations have been observed, e. g., by Gomez et al, [86] Mulqueen et al [87] and the authors of this review. [59] These effects can be tentatively attributed to the particular electron configurations of these ions and other highly complex properties of transition elements (polarisability, relativistic effects, anisotropic binding to biomolecules). A detailed discussion of these effects is beyond the scope of this review.…”

Synchrotron Scattering Methods for Nanomaterials and Soft Matter Research

“…A similar approach has been employed to unravel the combined effects of ionic strength, temperature, and pressure on protein-protein interaction potential and the phase behavior in dense lysozyme solutions [31]. A recent SAXS investigation probed the evolution of protein-protein interactions and liquid-liquid phase separation induced by trivalent salts and temperature in concentrated bovine serum albumin (BSA) solutions [32]. Proteins immobilized on polyelectrolyte brushes is another topic studied by SAXS, which enabled quantitative estimation of the concentration and location of adsorbed proteins within the brush layer [33,34].…”

A multi-channel in situ light scattering instrument utilized for monitoring protein aggregation and liquid dense cluster formation

“…As LDC formation obviously has also a high impact in biochemistry and biotechnology research, a variety of recent experiments has further investigated formation, dynamics, stability and symmetry of LDCs and related assemblies [6, 7, 8] and has also unveiled that various membrane-less organelles are highly dynamic dense liquids formed by a first order phase transition [3, 9]. Furthermore, “super-resolution” fluorescence and electron microscopy as well as X-ray scattering have identified macromolecule LDC formation in the presence of a variety of polymers and multivalent ions [10, 11, 12, 13]. In this context the flexibility of low-complexity protein domains and RNAs was recognized to promote the formation of LDCs [1, 2].…”