Optical characterization of molecular interaction strength in protein condensates

Abstract

Biomolecular condensates have recently been identified as a ubiquitous means of intracellular organization. Investigating the molecular interactions determining the formation, and physical properties of biomolecular condensates provides key insights for understanding their biological function, and dysfunction. Here, we applied Brillouin microscopy and quantitative phase imaging to quantify average molecular interaction strength, dry mass density, and protein volume fraction in protein condensates in vitro. We monitored the physical changes in FUS condensates in response to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and dry mass density. In contrast to solidification by chemical crosslinking, physical aging of condensates had only a small impact on the Brillouin shift. Physical aging was suppressed at a high ion concentration. Finally, we characterized sequence variations of the low-complexity domain of hnRNPA1 that change the driving force for phase separation and found that they also alter the physical properties of the condensates. Our results provide a new experimental perspective on the physical properties of protein condensates and their sensitivity to solution conditions, sequence, and as a function of time.