Hasan Cinar’s research while affiliated with TU Dortmund University and other places

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.

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.

A binary protein condensate that mimics postsynaptic densities revealed high pressure sensitivity. This may help to gain a better understanding of the biophysical causes of pressure‐related neurological disorders, such as the high‐pressure neurological syndrome experienced by deep‐sea divers. More information can be found in the Full Paper by R. Winter, H. S. Chan et al. (DOI: 10.1002/chem.201905269).

Interactions 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.

Biomolecular condensates consisting of proteins and nucleic acids can serve critical biological functions, so that some condensates are referred as membraneless organelles. They can also be disease‐causing, if their assembly is misregulated. A major physicochemical basis of the formation of biomolecular condensates is liquid–liquid phase separation (LLPS). In general, LLPS depends on environmental variables, such as temperature and hydrostatic pressure. The effects of pressure on the LLPS of a binary SynGAP/PSD‐95 protein system mimicking postsynaptic densities, which are protein assemblies underneath the plasma membrane of excitatory synapses, were investigated. Quite unexpectedly, the model system LLPS is much more sensitive to pressure than the folded states of typical globular proteins. Phase‐separated droplets of SynGAP/PSD‐95 were found to dissolve into a homogeneous solution already at ten‐to‐hundred bar levels. The pressure sensitivity of SynGAP/PSD‐95 is seen here as a consequence of both pressure‐dependent multivalent interaction strength and void volume effects. Considering that organisms in the deep sea are under pressures up to about 1 kbar, this implies that deep‐sea organisms have to devise means to counteract this high pressure sensitivity of biomolecular condensates to avoid harm. Intriguingly, these findings may shed light on the biophysical underpinning of pressure‐related neurological disorders in terrestrial vertebrates.

We 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.

Biological functions of proteins are underpinned by physicochemical processes governed by the intra‐ and intermolecular interactions among proteins and other biomolecules. Understanding these interactions is important not only for deciphering normal biological functions but also for gaining insights into disease processes as well as for biotechnological applications. Liquid–liquid phase separations (LLPSs) of proteins, which play a critical role in membrane‐less compartmentalization of the intra‐organismal space, are typically more sensitive to pressure than the folding of proteins, suggesting that organisms thriving in the deep sea have to mitigate this pressure sensitivity. Particular osmolytes, such as TMAO, are able to stabilize protein condensates under pressure. In their Review article on page 13049 ff., H. S. Chan, R. H. A. Winter et al. discuss recent experimental findings and present an outline of the basic thermodynamics of temperature‐, pressure‐, and osmolyte‐dependent LLPSs.

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.

Biomolecular condensates can be functional (e.g., as "membrane-less organelles") or dysfunctional (e.g., as precursor to pathological protein aggregates). A major physical underpinning of biomolecular condensates is liquid-liquid phase separation (LLPS) of proteins and nucleic acids. Here we investigate the effects of temperature and pressure on the LLPS of the eye-lens protein γ-crystallin, using UV/Vis absorption, fluorescence and light microscopy to characterize the mesoscopic phase states. Quite unexpectedly, the LLPS of γ-crystallin is much more sensitive to pressure than folded states of globular proteins. At low temperatures, the phase-separated droplets of γ-crystallin dissolve into a homogeneous solution at as low as ~0.1 kbar whereas proteins typically unfold above ~3 kbar. This observation suggests, in general, that organisms thriving at high-pressure conditions in the deep sea, with pressure up to 1 kbar, have to cope with this pressure-sensitivity of biomolecular condensates to avoid detrimental impact on their physiology. Interestingly, our experiments demonstrate that trimethylamine-N-oxide, an osmolyte upregulated in deep-sea fish, significantly enhances the stability of the condensed protein droplets, pointing to a previously unrecognized aspect of the adaptive advantage of increased concentrations of osmolytes in deep-sea organisms. As the birth place of life on Earth could have been the deep sea, studies of pressure effects on LLPS as presented here are relevant to the possible formation of protocells under prebiotic conditions. A physical framework to conceptualize our observations and further ramifications of biomolecular LLPS under low temperatures and high hydrostatic pressures are discussed.