Impact of organic osmolytes and crowding on the protein-protein interaction at high pressures

Abstract

Applying small-angle X-ray scattering in combination with liquid-state theory, 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 and in presence of natural osmolyte mixtures occurring within cells of organisms that thrive under extreme environmental conditions. The deep-sea osmolyte trimethylamine-N-oxide (TMAO) is shown to play a crucial and singular role in its ability to not only guarantee sustainability of the native protein’s folded state under harsh environmental conditions, but it also controls water-mediated intermolecular interactions at high pressure, thereby preventing contact formation and hence aggregation of proteins. 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 semi-dilute 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. Further, the study on the effects of cosolvents and crowding agents on the pressure-induced conformational changes of the dimeric enzyme horse liver alcohol dehydrogenase (LADH) on the quaternary, secondary and tertiary structural level reveal that oligomerization and subunit dissociation are modulated by cosolvents such as urea or TMAO as well as by the crowding agent PEG, based on their tendency to bind to the protein’s interface or act via the excluded volume effect, respectively.