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High pressure increases the propensity of cationic antimicrobial peptides to form active helical structures on bacterial membranes, indicating that high-pressure could boost cAMP activity in high-pressure food processing.

The high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid-liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity.

The chaperonin system GroEL-GroES is present in all kingdoms of life and rescues proteins from improper folding and aggregation upon internal and external stress conditions, including high temperatures and pressures. Here, we set out to explore the thermo- and piezostability of GroEL, GroES and the GroEL-GroES complex in the presence of cosolvents, nucleotides and salts employing quantitative FTIR spectroscopy and small-angle X-ray scattering. Owing to its high biological relevance and lack of data, our focus was especially on the effect of pressure on the chaperonin system. The experimental results reveal that the GroEL-GroES complex is remarkably temperature stable with an unfolding temperature beyond 70 °C, which can still be slightly increased by compatible cosolutes like TMAO. Conversely, the pressure stability of GroEL and hence the GroEL-GroES complex is rather limited and much less than that of monomeric proteins. Whereas GroES is pressure stable up to ∼5 kbar, GroEl and the GroEl-GroES complex undergo minor structural changes already beyond 1 kbar, which can be attributed to a dissociation-induced conformational drift. Quite unexpectedly, no significant unfolding of GroEL is observed even up to 10 kbar, however, i.e., the subunits themselves are very pressure stable. As for the physiological relevance, the structural integrity of the chaperonin system is retained in a relatively narrow pressure range, from about 1 to 1000 bar, which is just the pressure range encountered by life on Earth.

Tubulin is one of the main components of the cytoskeleton of eukaryotic cells. The formation of microtubules depends strongly on environmental and solution conditions, and has been found to be among the most pressure sensitive processes in vivo. We explored the effects of different types of cosolvents, such as trimethylamine-N-oxide (TMAO), sucrose and urea, and crowding agents to mimic cell-like conditions, on the temperature and pressure stability of the building block of microtubules, i. e. the α/β-tubulin heterodimer. To this end, fluorescence and FTIR spectroscopy, differential scanning and pressure perturbation calorimetry as well as fluorescence anisotropy and correlation spectroscopies were applied. The pressure and temperature of dissociation of α/β-tubulin as well as the underlying thermodynamic parameters upon dissociation, such as volume and enthalpy changes, have been determined for the different solution conditions. The temperature and pressure of dissociation of the α/β-tubulin heterodimer and hence its stability increases dramatically in the presence of TMAO and the nanocrowder sucrose. We show that by adjusting the levels of compatible cosolutes and crowders, cells are able to withstand deteriorating effects of pressure even up to the kbar-range.

The application of co-solvents and high pressure has been shown to be an efficient means to modify the kinetics of enzyme-catalyzed reactions without compromising enzyme stability, which is often limited by temperature modulation. In this work, the high-pressure stopped-flow methodology was applied in conjunction with fast UV/Vis detection to investigate kinetic parameters of formate dehydrogenase reaction (FDH), which is used in biotechnology for cofactor recycling systems. Complementary FTIR spectroscopic and differential scanning fluorimetric studies were performed to reveal pressure and temperature effects on the structure and stability of the FDH. In neat buffer solution, the kinetic efficiency increases by one order of magnitude by increasing the temperature from 25° to 45 °C and the pressure from ambient up to the kbar range. The addition of particular co-solvents further doubled the kinetic efficiency of the reaction, in particular the compatible osmolyte trimethylamine-N-oxide and its mixtures with the macromolecular crowding agent dextran. The thermodynamic model PC-SAFT was successfully applied within a simplified activity-based Michaelis-Menten framework to predict the effects of co-solvents on the kinetic efficiency by accounting for interactions involving substrate, co-solvent, water, and FDH. Especially mixtures of the co-solvents at high concentrations were beneficial for the kinetic efficiency and for the unfolding temperature.

The use of cosolutes and high hydrostatic pressure has been described as an efficient means to modulate the stability of enzymes and their catalytic activity. Cosolvents and pressure can lead to increased reaction rates without compromising the stability of the enzyme. Inspired by the multi-component nature of the crowded cellular milieu of biological cells of piezophiles, we studied the combined effects of macromolecular crowding agents, different types of cosolvents and pressure in concert on a hydrolysis reaction catalyzed by α-chymotrypsin. We have seen that crowding agents and cosolvents can have very diverse effects on enzymatic activity. Addition of the deep-sea osmolyte trimethylamine-N-oxide displays by far the most positive effect on the catalytic efficiency, keff, of the reaction, which is even markedly enhanced at high pressures. Addition of the chaotropic agent urea leads to the reverse effect, and PEG and dextran as two representative crowding agents of a different nature show nearly similar values for keff compared to the pure buffer data. Such information may not only be relevant for understanding life processes in extreme environments, but also for the use of enzymes in industrial processing, which often requires harsh conditions as well., ChemSystemsChem;3(2)

Deep subsurface environments can harbour high concentrations of dissolved ions, yet we know little about how this shapes the conditions for life. We know even less about how the combined effects of high pressure influence the way in which ions constrain the possibilities for life. One such ion is perchlorate, which is found in extreme environments on Earth and pervasively on Mars. We investigated the interactions of high pressure and high perchlorate concentrations on enzymatic activity. We demonstrate that high pressures increase α-chymotrypsin enzyme activity even in the presence of high perchlorate concentrations. Perchlorate salts were shown to shift the folded α-chymotrypsin phase space to lower temperatures and pressures. The results presented here may suggest that high pressures increase the habitability of environments under perchlorate stress. Therefore, deep subsurface environments that combine these stressors, potentially including the subsurface of Mars, may be more habitable than previously thought., Gault et al. show that high pressures increase α-chymotrypsin enzyme activity in the presence of high perchlorate concentrations. These perchlorate salts shift the folded enzyme phase space to lower temperatures and pressure and may move the optimum enzyme activity towards lower temperatures in addition to higher pressures, which has implications for Martian habitability.