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This is a Quantitative Study study.

October 2, 2025

Fast Microdialysis Buffer Exchange to Study Poly(glutamate)/Lysozyme Coacervates in Concentrated Conditions

PBPierre-Louis BrassartCentre National de la Recherche Scientifique MCMaria da ConceiçãoAir Liquide (United States)AVAlina VashchukCentre National de la Recherche Scientifique

Key Points

Rapid buffer exchange enhances understanding of microvolumes in protein interactions, leading to novel insights.
Key data includes that ionic strength profoundly influences coacervation behavior, especially above 80 g/L lysozyme.
The use of a microdialysis chip provides a precise method to control experimental conditions in real-time.
Findings indicate that coacervates can transform from fluid-like to solid-like states, with distinct effects on diffusivity.

Abstract

Complex coacervation between oppositely charged proteins and polyelectrolytes has broad relevance, from membraneless organelles to biomedical applications. Unlike well-characterized synthetic polyelectrolytes, protein coacervates pose experimental challenges due to limited quantities, constraints of mild handling, fine-tuning of pH, and ionic strength affecting contributions of charge patches or non-Coulombic binding. To alleviate these constraints, we propose to use a microdialysis chip allowing in situ control of microvolumes. We studied model lysozyme/polyglutamate coacervates. Rapid equilibration with phosphate buffers of varying concentrations showed that (i) fine control of the ionic strength was essential due to the release of counterions affecting the phase diagram above 80 g/L lysozyme, (ii) phase separation was reversible, (iii) chirality of the polyglutamate affects coacervation, and (iv) coacervates can be annealed into homogeneous films allowing FRAP measurements across fluid-like to solid-like transition as betrayed by a marked decrease of polyglutamate diffusivity below a threshold of 60 mM phosphate.

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