Tuning biological processes via co-solutes: from single proteins to protein condensates - the case of α-elastin condensation

Abstract

Protein condensates as membrane-less compartments play a pivotal role in cellular processes. The stabilization of protein condensation can be tuned using cosolutes which directly impact biological function. In this study, we report the result of a rigorous study of the influence of cosolutes changes on hydration entropy and enthalpy upon condensate formation, by means of THz-calorimetry. Our results unveil quantitative insights into the fine tuning of the free energy imbalance, via hydrophobic/entropic and hydrophilic/enthalpic hydration which can result in cosolute-mediated stabilization or destabilization of protein condensates. These results shed new light on the regulatory potential of co-solutes within cells, to tune Liquid-Liquid Phase Separation (LLPS). Furthermore, we demonstrate the transferability of the underlying molecular concepts of cosolute addition to two fundamental biological processes: protein folding and denaturation. This study provides a blueprint for controlled modulating LLPS via cosolute additions, with promising implications in both biological and medical applications.

Fig. 1. ATR sedimentation assay for measuring THz spectra upon LLPS. (Left) Schematics illustrating the formation of protein condensates upon LLPS and their sedimentation on the ATR crystal during the measurements. (Right) The measured absorption (Δα) as a function of time (for one example), plotted as a difference with respect to the diluted protein phase (initial spectrum). During the time-series, the sedimentation of protein condensates causes the observed changes in the THz features associated with cavity-wrap (hydrophobic, red) and bound water (hydrophilic, blue) hydration contributions, as discussed in the text.

Fig. 2. The addition of NaCl affects the release of cavity-wrap hydration water and the retention of hydrogen bound water. (A) Difference THz spectra upon LLPS as a function of increasing cosolute concentrations, shown for NaCl. The changes in cavity-wrap and bound water features are highlighted in red and blue, respectively. These changes are quantified using Δα_wrap and shown in the plot and defined in the text. (B) The plot shows ΔΔα_wrap and for three different cosolute concentrations. (C) Changes in ΔΔH_bound and −TΔΔS_wrap solvation upon the addition of NaCl. ΔΔH_bound and −TΔΔS_wrap are the partial contributions to hydrophilic and hydrophobic hydration as deduced by means of THz-calorimetry, see methods and eqn (5) and (6).

Fig. 3. The addition of a co-solute can favor (green quadrant) or disfavor (orange quadrant) liquid–liquid phase separation. The plot summarizes the result of THz calorimetry for changes with respect to hydration entropy (from hydrophobic hydration), plotted on the x-axis, and hydration enthalpy (from hydrophilic hydration), plotted on the y-axis, upon LLPS when adding co-solutes. The symbols and colors represent the choice of cosolute and their variation with respect to concentration and temperatures, see the legend.

Fig. 4. Bridging the cosolute effect from single proteins to protein condensates. The plot summarizes the three classes of cosolutes as found in our THz-calorimetry study, based on their effect on LLPS (i.e. on the stability of protein condensates). The classification matches the one known for the cosolute effect on protein stability. As discussed in the text, a parallelism can be drawn between the two fields, despite the distinct hydration properties of proteins and condensates.