Liquid–liquid phase separation (LLPS) of proteins and other biomolecules play a critical role in the organization of extracellular materials and membrane‐less compartmentalization of intra‐organismal ...spaces through the formation of condensates. Structural properties of such mesoscopic droplet‐like states were studied by spectroscopy, microscopy, and other biophysical techniques. The temperature dependence of biomolecular LLPS has been studied extensively, indicating that phase‐separated condensed states of proteins can be stabilized or destabilized by increasing temperature. In contrast, the physical and biological significance of hydrostatic pressure on LLPS is less appreciated. Summarized here are recent investigations of protein LLPS under pressures up to the kbar‐regime. Strikingly, for the cases studied thus far, LLPSs of both globular proteins and intrinsically disordered proteins/regions are typically more sensitive to pressure than the folding of proteins, suggesting that organisms inhabiting the deep sea and sub‐seafloor sediments, under pressures up to 1 kbar and beyond, have to mitigate this pressure‐sensitivity to avoid unwanted destabilization of their functional biomolecular condensates. Interestingly, we found that trimethylamine‐N‐oxide (TMAO), an osmolyte upregulated in deep‐sea fish, can significantly stabilize protein droplets under pressure, pointing to another adaptive advantage for increased TMAO concentrations in deep‐sea organisms besides the osmolyte's stabilizing effect against protein unfolding. As life on Earth might have originated in the deep sea, pressure‐dependent LLPS is pertinent to questions regarding prebiotic proto‐cells. Herein, we offer a conceptual framework for rationalizing the recent experimental findings and present an outline of the basic thermodynamics of temperature‐, pressure‐, and osmolyte‐dependent LLPS as well as a molecular‐level statistical mechanics picture in terms of solvent‐mediated interactions and void volumes.
Protein–protein interactions: In this Review, we show that protein condensates are typically more sensitive to pressure than the folding of proteins, suggesting that organisms thriving in the deep sea must mitigate this pressure sensitivity.
Biomolecular assembly processes based on liquid-liquid phase separation (LLPS) are ubiquitous in the biological cell. To fully understand the role of LLPS in biological self-assembly, it is necessary ...to characterize also their kinetics of formation and dissolution. Here, we introduce the pressure-jump relaxation technique in concert with UV/Vis and FTIR spectroscopy as well as light microscopy to characterize the evolution of LLPS formation and dissolution in a time-dependent manner. As a model system undergoing LLPS we used the globular eye-lens protein γD-crystallin. As cosolutes and macromolecular crowding are known to affect the stability and dynamics of biomolecular condensates in cellulo, we extended our kinetic study by addressing also the impact of urea, the deep-sea osmolyte trimethylamine-N-oxide (TMAO) and a crowding agent on the transformation kinetics of the LLPS system. As a prerequisite for the kinetic studies, the phase diagram of γD-crystallin at the different solution conditions also had to be determined. The formation of the droplet phase was found to be a very rapid process and can be switched on and off on the 1-4 s timescale. Theoretical treatment using the Johnson-Mehl-Avrami-Kolmogorov model indicates that the LLPS proceeds via a diffusion-limited nucleation and growth mechanism at subcritical protein concentrations, a scenario which is also expected to prevail within biologically relevant crowded systems. Compared to the marked effect the cosolutes take on the stability of the LLPS region, their effect at biologically relevant concentrations on the phase transformation kinetics is very small, which might be a particular advantage in the cellular context, as a fast switching capability of the transition should not be compromised by the presence of cellular cosolutes.
The free energy and conformational landscape of biomolecular systems as well as biochemical reactions depend not only on temperature and pressure, but also on the particular solution conditions. Such ...conditions include the effects of cosolvents (for example osmolytes) and macromolecular crowding, which are crucial components to understand the energetics and kinetics of biological processes in living system. Such conditions are also important for the understanding of many debilitating diseases, such as those where misfolding and amyloid formation of proteins are involved. Moreover, understanding their effects on biomolecular processes is prerequisite for designing industrially relevant enzymatic reactions, which seldom take place under neat conditions. Here, we review and discuss experimental and theoretical studies on the characterization of cosolvent and crowding induced effects in biologically relevant systems, approaching even the complexity of living organisms. In particular, we focus on cosolvent and crowding effects on the conformational equilibrium and folding kinetics of proteins and nucleic acids as well as on enzymatic reactions, including their effects on the temperature and pressure dependence of these processes. By presenting a few representative examples, we show how such effects are unveiled and described in thermodynamic and kinetic terms.
The high content of macromolecules and cosolvents inside cells can significantly modulate the energy landscape of biomolecular systems and reactions. Effects induced by the cellular milieu as a solvent are reviewed.
Recently, 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.
High hydrostatic pressure affects the structure, dynamics, and stability of biomolecular systems and is a key parameter in the context of the exploration of the origin and the physical limits of ...life. This review lays out the conceptual framework for exploring the conformational fluctuations, dynamical properties, and activity of biomolecular systems using pressure perturbation. Complementary pressure-jump relaxation studies are useful tools to study the kinetics and mechanisms of biomolecular phase transitions and structural transformations, such as membrane fusion or protein and nucleic acid folding. Finally, the advantages of using pressure to explore biomolecular assemblies and modulate enzymatic reactions are discussed.
Cold denaturation (including supercooling) and pressure perturbation are able to dissolve protein aggregates and, in some cases, amyloid fibrils (see picture). These studies provide additional ...details on the polymorphic forms of amyloid structures and their precursors as well as the transformation processes between polymorphic states.
Knowledge of the intermolecular interaction potential of proteins as a function of their solution conditions is essential for understanding protein aggregation, crystallization, and the phase ...behavior of proteins in general. Here, we report on a combined small-angle X-ray scattering and liquid-state theoretical approach to study dense lysozyme solutions as a function of temperature and pressure, but also in the presence of salts and osmolytes of different nature. We show that the pressure-dependent interaction potential of lysozyme changes in a nonlinear fashion over a wide range of temperatures, salt and protein concentrations, indicating that changes of the bulk water structure mediate the pressure dependence of the intermolecular forces. We present also results on the effect of high hydrostatic pressure on the phase behavior of dense lysozyme solutions in the liquid-liquid phase-coexistence region. As also shown in this study, the application of pressure can be used to fine-tune the second virial coefficient of protein solutions, which can be used to control nucleation rates and hence protein crystallization, or to prevent protein aggregation. Moreover, these results are also important for understanding the hydration behavior of biological matter under extreme environmental conditions, and the high stability of dense protein solutions (as they occur intracellularly) in organisms thriving under hydrostatic pressure conditions such as in the deep sea, where pressures up to the 100 MPa-level are reached.
The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the ...self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most in vitro studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a--consequently--retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.
Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee
. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits ...antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-
-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-
-glycero-3-
-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.
The effect of an amyloidogenic intrinsically disordered protein, α‐synuclein, which is associated with Parkinson's disease (PD), on the conformational dynamics of a DNA hairpin (DNA‐HP) was studied ...by employing the single‐molecule Förster resonance energy transfer method. The open‐to‐closed conformational equilibrium of the DNA‐HP is drastically affected by binding of monomeric α‐synuclein to the loop region of the DNA‐HP. Formation of a protein‐bound intermediate conformation is fostered in the presence of an aqueous two‐phase system mimicking intracellular liquid‐liquid phase separation. Using pressure modulation, additional mechanistic information about the binding complex could be retrieved. Hence, in addition to toxic amyloid formation, α‐synuclein may alter expression profiles of disease‐modifying genes in PD. Furthermore, these findings might also have significant bearings on the understanding of the physiology of organisms thriving at high pressures in the deep sea.
The effect of an amyloidogenic intrinsically disordered protein, α‐synuclein, on the conformational dynamics of a DNA hairpin was studied by employing the single‐molecule Förster resonance energy transfer method. Using pressure modulation, additional mechanistic information about the binding complex could be retrieved.
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