Phase behavior of lysozyme solutions in the liquid–liquid phase coexistence region at high hydrostatic pressures

Abstract

We present results from small-angle X-ray scattering and turbidity measurements on the effect of high hydrostatic pressure on the phase behavior of dense lysozyme solutions in the liquid-liquid phase separation region, and characterize the underlying intermolecular protein-protein interactions as a function of temperature and pressure under charge-screening conditions (0.5 M NaCl). A reentrant liquid-liquid phase separation region is observed at elevated pressures, which may originate in the pressure dependence of the solvent-mediated protein-protein interaction. A temperature-pressure-concentration phase diagram was constructed for highly concentrated lysozyme solutions over a wide range of temperatures, pressures and protein concentrations including the critical region of the liquid-liquid miscibility gap.

Temperature, Hydrostatic Pressure, and Osmolyte Effects on Liquid–Liquid Phase Separation in Protein Condensates: Physical Chemistry and Biological Implications

“…[25,47,48] The data presented below were obtainedu sing FTIR and CD spectroscopy,t urbidity measurements, and phase-contrast light and fluorescencem icroscopy in conjunction with variousd evices for high-pressure measurements to study the structurea nd phase properties of the proteins over aw ide range of temperatures, pressures and osmolyte concentrations (see refs. [42,44,[76][77][78] for experimental details). Using lysozyme, g-crystallin, a-elastin, and an N-terminal IDR of Ddx4 as examples, representative resultsf or different kinds of protein systemsa re highlighted and general conceptual frameworks for rationalizing the observed T-, p-, and osmolyte-dependence of protein LLPS are explored.…”

“…Upon pressurization at 18 8C, at 40 and 250 bar,r espectively,t he sample is observed to cross the phase boundary out of the LLPS regioni nto ah omogenous one-phase region and then enters ah igh-pressureL LPS region (HP-LLPS) again at ah igher pressure. [42,77] In other words, the system exhibits ap ressure-induced re-entrant phase transition (RPT). From the combined temperature andp ressure dependent data, a p-T phase diagram was constructed for the LLPS of lysozyme [77] (Figure3b).…”

“…It turned out to be also an ideal model system for experimental and theoretical investigations of intermolecular interactions and phase behavior under high hydrostatic pressures. As determined by high‐pressure small‐angle scattering (SAXS) and FTIR spectroscopy, the structure of lysozyme does not undergo discernible change up to 4 kbar at temperatures below the unfolding temperature at pH 7 (∼72 °C at 1 bar), indicating that the protein remains essentially in its native, folded conformation . Accordingly, the protein is taken to retain its native folded structure in the LLPS region covered by the experimental temperatures and pressures discussed in the following and can be treated essentially as a rigid object.…”

Pressure‐Induced Dissolution and Reentrant Formation of Condensed, Liquid–Liquid Phase‐Separated Elastomeric α‐Elastin

“…[2] A large fraction of the globalb iosphere's organisms (> 60 %) is thriving at ad epth in excess of 1000 ma nd therefore exposed to pressureso f1 00 bar andm ore, reaching even the 1kbar (100 MPa) level at the sea floor in the Marianat rench. [4,5] Pressure perturbation re-distributes populations of conformational states by virtue of the states' different volumes, with increasing pressure favoringt he state with the lowest partial molar volume. [3] Furthermore, pressure can be used to fine-tune the intermoleculari nteraction potentialb etween proteins and thus serve as an important probe for mapping the conformational landscapeo fb iomolecules.…”

“…[3] Furthermore, pressure can be used to fine-tune the intermoleculari nteraction potentialb etween proteins and thus serve as an important probe for mapping the conformational landscapeo fb iomolecules. [4,5] Pressure perturbation re-distributes populations of conformational states by virtue of the states' different volumes, with increasing pressure favoringt he state with the lowest partial molar volume. As the partial molar volumeo fb iomolecules is sensitive to hydration changes,p ressure studies can providec rucial insights into both the packing and hydrational properties of biomolecules.…”