Viscoelasticity of globular protein-based biomolecular condensates

Abstract

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.

Fig. 1. Schematic illustrating the complex coacervates evaluated herein. Coacervates between polyK (degree of polymerization (DP) = 30) and three proteins with increasing disordered peptide content were compared to a similar coacervate prepared with polyK and polyD (DP = 30). The protein structures show negative (red) and positive (blue) residue locations for iso-GFP, tag6-GFP and tag12-GFP (left to right). Polypeptide structures were predicted using PEP-FOLD3, while protein structures were predicted using AlphaFold. Optical microscopy images show the formation of liquid-like droplets at the optimal mixing ratio for phase separation.

Fig. 2. Phase diagrams with GFP and polyK. (A) Iso-GFP, tag6-GFP, and tag12-GFP turbidity as a function of sodium chloride concentration and charge fraction (f⁺) calculated as M⁺/(M⁺ + M⁻) where M⁺ and M⁻ are the charge per polymer and protein, respectively. GFP 40 µM, Tris (10 mM, pH 7.4). (B) Brightfield images of GFP coacervates at varying sodium chloride concentrations and charge fraction. Scale bar 10 µm.

Fig. 3. Video particle tracking in model condensates. (A) Mean squared displacement as a function of lag time for iso-GFP (blue), tag6-GFP (purple) and tag12-GFP (maroon) with polyK (f⁺ = 0.7) (Tris 10 mM, NaCl 100 mM). Plots depict average of three independent measurements. Shaded areas show standard deviation. Inset: single frame from particle tracking video of iso-GFP–polyK (f⁺ = 0.9) condensate with 500 nm beads embedded. Scale bar is 20 µm. (B) Viscosity for iso-GFP (blue), tag6-GFP (purple), and tag12-GFP (maroon) condensates when prepared at 100 mM NaCl with 40 µM protein and different polyK concentrations. Iso-GFP coacervates were prepared with 6 polyK concentrations: 10, 20, 40, 80, 148, 500 µM, corresponding to f⁺ = 0.38, 0.55, 0.71, 0.83, 0.9 and 0.97. Similarly, tag6-GFP coacervates were prepared with polyK at 40, 80 and 148 µM and tag12-GFP with polyK at 20, 40, 80 and 148 µM. Three separate measurements (data points), average (black line), and standard deviation (error bars), are shown. Enlarged portion of plot displays viscosity of polyK–polyD coacervates (grey), coacervates were prepared at 3 polyK concentrations 30, 40 and 80 µM, corresponding to charge ratios f⁺ = 0.43, 0.5, and 0.67.

Fig. 4. Viscoelasticity of GFP–polyK condensates. (A) Mean square displacement (MSD) of iso-GFP coacervates (polyK 40 µM) determined from DLS microrheology (grey) and MSD determined from video particle tracking microrheology (blue). Background colored regions qualitatively illustrate different timescales. (B) Complex modulus of iso-GFP coacervate from DLS microrheology. G′ solid line, G″ dashed line. Grey lines represent two-component Maxwell fit. (C) Schematic illustrating protein polymer behavior at different timescales. At longest time scales (blue background) proteins and polymers can flow. At intermediate times (white background) behavior is dominated by interactions between charged residues on GFP and polyK. At short timescales (green background) residue interactions with water dominate.

Fig. 5. Condensate rheological properties as a function of mixing ratio. (A) Complex modulus of iso-GFP (40 µM) and polyK coacervates at different charge ratios (f⁺ = 0.55, 0.71, 0.90), (B) tag6-GFP (40 µM) and polyK at different charge ratios (f⁺ = 0.71, 0.90), (C) tag12-GFP (40 µM) and polyK at different charge ratios (f⁺ = 0.55, 0.71). (D) Viscosity (η₀ = 2G₀τ₀) from DLS (filled circles) and VPT (empty circles) for iso-GFP (40 µM) and polyK coacervates at different charge ratios (f⁺ = 0.55, 0.71, 0.90) (blue); tag6-GFP (40 µM) and polyK at different charge ratios (f⁺ = 0.71, 0.90) (purple), tag12-GFP (40 µM) with polyK at different charge ratios (f⁺ = 0.55, 0.71) (red) and polyD30 (f⁺ = 0.50) (grey). (E) Plateau modulus (G_N) of iso-GFP (40 µM) and polyK coacervates at charge ratios f⁺ = 0.55, 0.71, 0.90 (blue), tag12-GFP (40 µM) with polyK at charge ratios f⁺ = 0.71, 0.90 (purple), tag12-GFP (40 µM) with polyK at charge ratios f⁺ = 0.55, 0.71 (red) and polyD30 (f⁺ = 0.50) (grey). (F) Complex modulus of iso-GFP (blue) (40 µM) with polyK (40 µM, f⁺ = 0.71) in comparison to polyD (grey) (40 µM) with polyK (40 µM, f⁺ = 0.5).