Macromolecular condensation buffers intracellular water potential

Abstract

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of ‘structured’ water molecules within their hydration layers, reducing the available ‘free’ bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

Protein structure–function continuum model: Emerging nexuses between specificity, evolution, and structure

“…The importance of water activity under low water conditions in vitro was proposed by Halling (1994) and is now well accepted. Watson et al (2023) in a just‐published paper have focused on its importance in vivo under the crowded intracellular conditions. Zaslavsky and Uversky (2018) had earlier pointed out how LLPS can affect the availability of water for cellular needs.…”

How plants sense and respond to osmotic stress

“…Moreover, this condensed phase coexists with the dilute phase of macromolecules. One hypothesis is that the formation and dissolution of biomolecular condensates free and integrate “structured” water, quickly counteracting osmotic perturbation of cytosolic solutions so that biochemical reactions occur (Watson et al, 2023). However, this hypothesis may require a prerequisite that the plasma membrane becomes water impermeable as the formation of biomolecular condensates elevates osmotic potential, although it might reduce matric potential.…”

“…As a result, hyperosmotic stress increases molecular crowding of intracellular abundant molecules, such as proteins, nucleic acids, lipids, and metabolites. Whether fluid volume reduction or molecular crowding themselves can be a signal input upon hyperosmotic stress has been discussed for many years (Minton et al, 1992;Fiol and Kultz, 2007;Hoffmann et al, 2009;Kultz, 2012), and has become a hot topic due to progress in biomolecular condensates both in animals and plants (Majumder and Jain, 2020;Cuevas-Velazquez et al, 2021;Dorone et al, 2021;Boyd-Shiwarski et al, 2022;Wang et al, 2022a;Watson et al, 2023). Cells have many membrane-less organelles of mesoscopic size (approximately 0.2-2 μm) such as the nucleolus, Cajal bodies, nuclear stress bodies, processing bodies (P-bodies), and stress granules that are related to translational repression and/or mRNA decay upon environmental changes, especially hyperosmotic stress (Cuevas-Velazquez and Dinneny, 2018;Luo et al, 2018;Emenecker et al, 2020Emenecker et al, , 2021Lee and Martienssen, 2021;Lei et al, 2021;Xu et al, 2021;Allen and Strader, 2022;Hirose et al, 2023).…”

“…However, this hypothesis may require a prerequisite that the plasma membrane becomes water impermeable as the formation of biomolecular condensates elevates osmotic potential, although it might reduce matric potential. Because the ability to undergo LLPS may be a universal property of macromolecules and numerous proteins tend to phase separate in response to macromolecular crowding (Alberti et al, 2019; Watson et al, 2023; Zhang et al, 2023b), the physiological relevance of phase separation in cells should be carefully interpreted.…”

DNA Framework–Programmed Nanoscale Enzyme Assemblies

“…The programmability and addressability of DNA frameworks provide remarkable flexibility in constructing multienzyme assemblies, enabling precise control over their directional location, number, and distance. − Recently, various well-defined enzyme assemblies have been constructed using DNA origami and DNA tile nanostructures. − Nevertheless, a major challenge remains in the three-dimensional (3D) programming of enzyme assemblies with defined activity and spatial arrangement in vitro. − Herein, we present an enzyme cascade enhancement strategy based on a tetrahedral DNA framework (TDF) encoding system providing tunable 3D spatial topological configurations and identical edge-to-edge distances for programming nanoscale enzyme assemblies (Figure a). We demonstrate that TDF encoded with single-strand DNA (ssDNA) serves as a regular scaffold for creating multienzyme assemblies with different stoichiometric ratios.…”