Modulation of protein-saccharide interactions by deep-sea osmolytes under high pressure stress.

Deep-sea organisms must cope with high hydrostatic pressures (HHP) up to the kbar regime to control their biomolecular processes. To alleviate the adverse effects of HHP on protein stability most organisms use high amounts of osmolytes. Little is known about the effects of these high concentrations on ligand binding. We studied the effect of the deep-sea osmolytes trimethylamine-N-oxide, glycine, and glycine betaine on the binding between lysozyme and the tri-saccharide NAG3, employing experimental and theoretical tools to reveal the combined effect of osmolytes and HHP on the conformational dynamics, hydration changes, and thermodynamics of the binding process. Due to their different chemical makeup, these cosolutes modulate the protein-sugar interaction in different ways, leading to significant changes in the binding constant and its pressure dependence. These findings suggest that deep-sea organisms may down- and up-regulate reactions in response to HHP stress by altering the concentration and type of the intracellular osmolyte.

Leveraging liquid-liquid phase separation and volume modulation to regulate the enzymatic activity of formate dehydrogenase.

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO2. Pressurization was found to lead to an increase of the binding affinity (decrease of KM, respectively) and a decrease of the turnover number, kcat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the KM-values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches.

Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View.

Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.

Biomolecular Condensates under Extreme Martian Salt Conditions.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

Exploring the polymorphism, conformational dynamics and function of amyloidogenic peptides and proteins by temperature and pressure modulation.

Our understanding of amyloid structures and the mechanisms by which disease-associated peptides and proteins self-assemble into these fibrillar aggregates, has advanced considerably in recent years. It is also established that amyloid fibrils are generally polymorphic. The molecular structures of the aggregation intermediates and the causes of molecular and structural polymorphism are less understood, however. Such information is mandatory to explain the pathological diversity of amyloid diseases. What is also clear is that not only protein mutations, but also the physiological milieu, i.e. pH, cosolutes, crowding and surface interactions, have an impact on fibril formation. In this minireview, we focus on the effect of the less explored physical parameters temperature and pressure on the fibrillization propensity of proteins and how these variables can be used to reveal additional mechanistic information about intermediate states of fibril formation and molecular and structural polymorphism. Generally, amyloids are very stable and can resist harsh environmental conditions, such as extreme pH, high temperature and high pressure, and can hence serve as valuable functional amyloid. As an example, we discuss the effect of temperature and pressure on the catalytic activity of peptide amyloid fibrils that exhibit enzymatic activity.

The multifaceted effects of DMSO and high hydrostatic pressure on the kinetic constants of hydrolysis reactions catalyzed by α-chymotrypsin.

The use of cosolvents and high hydrostatic pressure (HHP) 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 affecting the stability of the enzyme. Here, we studied the combined effects of one of the most used organic cosolvents, dimethyl sulfoxide (DMSO), and HHP to reveal their combined effect on the kinetic constants of an α-chymotrypsin-catalyzed peptide hydrolysis reaction. The Michaelis constant and the turnover number of the reaction respond differently to the two variables, and we observed an opposite effect of hydrostatic pressure and the dipolar cosolvent DMSO on the kinetic parameters. The results could be rationalized by determining the volume diagram of the reaction at the different solution conditions. In our case, the use of high hydrostatic pressure in concert with DMSO does not lead to an improvement of the enzymatic activity. However, the advantages of DMSO and HHP to increase the temperature stability of the enzyme and to increase the solubility of more hydrophobic substrates could still be useful.