1,721,033 research outputs found
Enabling High Activation of Glucose‐6‐Phosphate Dehydrogenase Activity Through Liquid Condensate Formation and Compression
No full text: Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles
The multifaceted effects of DMSO and high hydrostatic pressure on the kinetic constants of hydrolysis reactions catalyzed by α-chymotrypsin
No full textThe 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
Unraveling the binding characteristics of small ligands to telomeric DNA by pressure modulation
No full textRecently, non-canonical DNA structures, such as G-quadruplexes (GQs), were found to be highly pressure sensitive, suggesting that pressure modulation studies can provide additional mechanistic details of such biomolecular systems. Using FRET and CD spectroscopy as well as binding equilibrium measurements, we investigated the effect of pressure on the binding reaction of the ligand ThT to the quadruplex 22AG in solutions containing different ionic species and a crowding agent mimicking the intracellular milieu. Pressure modulation helped us to identify the different conformational substates adopted by the quadruplex at the different solution conditions and to determine the volumetric changes during complex formation and the conformational transitions involved. The magnitudes of the binding volumes are a hallmark of packing defects and hydrational changes upon ligand binding. The conformational substates of the GQ as well as the binding strength and the stoichiometry of complex formation depend strongly on the solution conditions as well as on pressure. High hydrostatic pressure can also impact GQs inside living cells and thus affect expression of genetic information in deep sea organisms. We show that sub-kbar pressures do not only affect the conformational dynamics and structures of GQs, but also their ligand binding reactions
Harnessing Pressure-Axis Experiments to Explore Volume Fluctuations, Conformational Substates, and Solvation of Biomolecular Systems
No full text: Intrinsic thermodynamic fluctuations within biomolecules are crucial for their function, and flexibility is one of the strategies that evolution has developed to adapt to extreme environments. In this regard, pressure perturbation is an important tool for mechanistically exploring the causes and effects of volume fluctuations in biomolecules and biomolecular assemblies, their role in biomolecular interactions and reactions, and how they are affected by the solvent properties. High hydrostatic pressure is also a key parameter in the context of deep-sea and subsurface biology and the study of the origin and physical limits of life. We discuss the role of pressure-axis experiments in revealing intrinsic structural fluctuations as well as high-energy conformational substates of proteins and other biomolecular systems that are important for their function and provide some illustrative examples. We show that the structural and dynamic information obtained from such pressure-axis studies improves our understanding of biomolecular function, disease, biological evolution, and adaptation
Deep sea osmolytes in action: their effect on protein-ligand binding under high pressure stress
No full textBecause organisms living in the deep sea and in the sub-seafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding reactions, must be adjusted to keep the associated volume changes low in order to function efficiently. Almost all organisms use organic cosolvents (osmolytes) to protect their cells from adverse environmental conditions. They counteract osmotic imbalance, stabilize the structure of proteins and maintain their function. We studied the binding properties of the prototypical ligand proflavine to two serum proteins with different binding pockets, BSA and HSA, in the presence of two prominent osmolytes, trimethylamine-N-oxide (TMAO) and glycine betaine (GB). TMAO and GB play an important role in the regulation and adaptation of life in deep-sea organisms. To this end, pressure dependent fluorescence spectroscopy was applied, supplemented by circular dichroism (CD) spectroscopy and computer modeling studies. The pressure-dependent measurements were also performed to investigate the intimate nature of the complex formation in relation to hydration and packing changes caused by the presence of the osmolytes. We show that TMAO and GB are able to modulate the ligand binding process in specific ways. Depending on the chemical make-up of the protein's binding pocket and thus the thermodynamic forces driving the binding process, there are osmolytes with specific interaction sites and binding strengths with water that are able to mediate efficient ligand binding even under external stress conditions. In the binding of proflavine to BSA and HSA, the addition of both compatible osmolytes leads to an increase in the binding constant upon pressurization, with TMAO being the most efficient, rendering the binding process also insensitive to pressurization even up to 2 kbar as the volume change remains close to zero. This effect can be corroborated by the effects the cosolvents impose on the strength and dynamics of hydration water as well as on the conformational dynamics of the protein
Modulation of enzymatic activity by aqueous two-phase systems and pressure - rivalry between kinetic constants
No full textWe studied the combined effects of an aqueous two-phase system (ATPS) invoking liquid-liquid phase separation and pressure on an enzymatic hydrolysis reaction. We show that simple steric crowding effects are not able to explain the kinetic constants and their pressure dependence in the ATPS. Additional contributions, such as changes in water activity and non-specific weak interactions with ATPS components have to be invoked to explain the results obtained. The findings are relevant for understanding cellular processes of piezophiles and might have significant bearings on biotechnological applications using liquid-liquid phase separation and pressure in concert for modulating enzymatic reactions
Mechanistic Insights into the c-MYC G-Quadruplex and Berberine Binding inside an Aqueous Two-Phase System Mimicking Biomolecular Condensates
No full text: We investigated the binding between the c-MYC G-quadruplex (GQ) and berberine chloride (BCl) in an aqueous two-phase system (ATPS) with 12.3 wt % polyethylene glycol and 5.6 wt % dextran, mimicking the highly crowded intracellular biomolecular condensates formed via liquid-liquid phase separation. We found that in the ATPS, complex formation is significantly altered, leading to an increase in affinity and a change in the stoichiometry of the complex with respect to neat buffer conditions. Thermodynamic studies reveal that binding becomes more thermodynamically favorable in the ATPS due to entropic effects, as the strong excluded volume effect inside ATPS droplets reduces the entropic penalty associated with binding. Finally, the binding affinity of BCl for the c-MYC GQ is higher than those for other DNA structures, indicating potential specific interactions. Overall, these findings will be helpful in the design of potential drugs targeting the c-MYC GQ structures in cancer-related biocondensates
Biomolecular Condensates under Extreme Martian Salt Conditions
No full textBiomolecular 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
Counteraction of denaturant-induced protein unfolding is a general property of stabilizing agents
No full textDSC measurements on RNase A at neutral pH show that five stabilizing agents, namely trimethylamine
N-oxide, glucose, sucrose, betaine and sodium sulfate, can counteract the destabilizing action of urea,
sodium perchlorate, guanidinium chloride and guanidinium thiocyanate. This is an important finding
inferring that counteraction has a common physical origin, regardless of the chemical differences
among the stabilizing agents and among the destabilizing ones. A rationalization is provided grounded
on the following line of reasoning: (a) the decrease in solvent-excluded volume effect is the main
stabilizing contribution of the native state; (b) its magnitude increases on increasing the density of the
aqueous solution; (c) the density increases significantly in the ternary solutions containing water, a
stabilizing agent and a destabilizing one, as indicated by the present experimental data
Alteration of Protein Binding Affinities by Aqueous Two-Phase Systems Revealed by Pressure Perturbation
No full textInteractions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifunctional proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo
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