Dynamic light scattering microrheology unveils the impact of folded protein domains on biomolecular condensate viscoelasticity across multiple time scales.

The phase separation of biomolecules into biomolecular condensates has emerged as a ubiquitous cellular process. Understanding how intrinsically disordered protein sequence controls condensate formation and material properties has provided fundamental biological insights and led to the development of functional synthetic condensates. While these studies provide a valuable framework to understand subcellular organization via phase separation they have largely ignored the presence of folded domains and their impact on condensate properties. We set out to determine how the distribution of sticker interactions across a globular protein contributes to rheological properties of condensates and to what extent globular protein-containing condensates differ from those formed from two disordered components. We designed three variants of green fluorescent protein with different charge patterning and used dynamic light scattering microrheology to measure the viscoelastic spectrum of coacervates formed with poly-lysine over a timescale of 10⁻⁶ to 10 seconds, elucidating the response of protein condensates in this range for the first time. We further showed that the phase behavior and rheological characteristics of the condensates varied as a function of both protein charge distribution and polymer/protein ratio, behavior that was distinct to condensates formed with folded domains. Together, this work enhances our fundamental understanding of dynamic condensed biomaterials across biologically relevant length- and time-scales.

Turbidity Data: Absorbance measurements at 600 nm were performed on a Tecan Infinite M200 Pro plate reader. Samples were prepared in tissue culture-treated polystyrene 384-well plates (ThermoFisher).

Video particle tracking: Videos were collected on a Nikon Eclipse Ti inverted microscope an analysed MatLab using code from the Elbaum-Garfinkle lab, available on online at https://zenodo.org/record/6818910#.YsxnrnbMI2x

DLS rheology data: Correllation curves were collected using a Malvern Zetasizer Nano ZS (633 nm laser) running Zetasizer software (version 7.12) and operated in 173° non-invasive backscatter detection mode. Anaysis was performed analysed using Version: 0.0.21 of DLSuR published by P. C. Cai, et al., in Dynamic light scattering microrheology for soft and living materials. Soft Matter 17, 1929–1939 (2021) and available at https://dlsur.readthedocs.io/ and on GitHub at https://github.com/PamCai/DLSuR.