pH-Responsive Elastin-Like Polypeptide Designer Condensates

Abstract

Biomolecular condensates are macromolecular complexes formed by liquid-liquid phase separation. They regulate key biological functions by reversibly compartmentalizing molecules in cells, in a stimulus-dependent manner. Designing stimuli-responsive synthetic condensates is crucial for engineering compartmentalized synthetic cells that are able to mimic spatiotemporal control over the biochemical reactions. Here, we design and test a family of condensate-forming, pH-responsive elastin-like polypeptides (ELPs) that form condensates above critical pH values ranging between 4 and 7, for temperatures between 20 and at 37 °C. We show that the condensation occurs rapidly, in sharp pH intervals (ΔpH < 0.3). For eventual applications in engineering synthetic cell compartments, we demonstrate that multiple types of pH-responsive ELPs can form mixed condensates inside micron-sized vesicles. When genetically fused with enzymes, receptors, and signaling molecules, these pH-responsive ELPs could be potentially used as pH-switchable functional condensates for spatially controlling biochemistry in engineered synthetic cells.

Keywords: biomolecular condensates; elastin-like polypeptides; liquid−liquid phase separation; pH-responsive coacervation; synthetic cells.

Figure 1

(a) ELPs are water-soluble protein polymers (black) and can form condensates (yellow) if the temperature increases beyond its LCST, referred to as the transition temperature (T_t). ELPs have a general sequence (GXGVP)_n, where X can be any amino acid (except proline) and n is the length of the polymer. Upon reaching the T_t, ELPs desolvate and form condensate droplets that eventually coalesce into larger condensates. Typically condensation occurs within a temperature change of <3 °C. (b) Schematic plots based on MacKay et al. model of the phase behavior of recombinant pH-responsive ELPs (eq 1). pH-responsive ELPs (PREs) can be made by introducing guest residues with side groups that can protonate such as negatively charged glutamic acid (E; Glu) or positively charged histidine (H; His). The ∂T/∂pH is the steepest near the isoelectric point (pI) of the PRE that is determined by the identity of the charged guest residue composition. (c) Schematic plots of T_t versus pH for PREs where guest residue contains a combination of E and H at various ratios (with H/E always below 1), and thus various pI. Under the right conditions, these PREs can be engineered to condense at different pHs at constant temperature (see gray dashed line). (d) Schematic plot of T_t versus pH for PREs with a fixed number of E and H, but varying in hydrophobicity, h (Urry hydrophobicity index). At a fixed temperature, PREs can be engineered to condense at different pH values (gray dashed line).

Figure 2

Characterization of the pH dependence of PRE phase transitions. Polymer concentration 25 μM, buffer PBS¹⁰⁰ (pH > 6) or SBS¹⁰⁰ (pH < 6). (a) Turbidity of PRE-pI-3.2 at constant pH plotted against a temperature at 1 °C/min. Temperature-trigged condensation is rapid and usually takes ∼3 °C. Black dotted lines are sigmoidal fits used to determine temperature transition T_t at the inflection point. (b) T_t vs pH for the PRE-pI-x polymers with various values of x. The pH inflection point changes minimally with the increased pI (due to the higher ratio of His/Glu). A higher number of His residues decreased the T_t range, with the case of PRE-pI-5.7 demonstrating the limit. Just tuning Glu/His ratio in PREs is insufficient to realize PREs with strong and sharp pH-induced transitions.

Figure 3

(a) T_t vs pH profiles for PRE-h-x polymers. Solid lines are sigmoidal fits. Transition temperatures of the PRE-h-x designs qualitatively follow the Urry hydrophobicity (h), with the exception of PRE-h-36 that contains multiple tyrosines in the guest residue composition (Table 2). Dotted gray lines are 37 °C and room temperature (21 °C). (b) Zoom for temperatures 37 °C (top) and 21 °C (bottom).

Figure 4

Sharpness of pH transitions demonstrated by using PRE-h-46 as a fluorescent pH ON/OFF switch. (a) Schematic overview of PRE-h-46 labeled near the c-terminal with AT532 dye (red) and AT612Q quencher (blue), above and below the condensation threshold (pH_t). Condensation brings the dye and quencher in close proximity leading to fluorescence quenching and a decrease in the fluorescence signal (“OFF” state). (b) Top: images of a 2 μL droplet of 25 μM AT532-PRE-h-46 + AT612Q-PRE-h-46 in SBS¹⁰⁰ buffer on a UV transilluminator plate at pH 6 (“ON”) and pH 1 (“OFF”). Bottom: camera images of cuvettes: at pH 1, the fluorescence completely disappears, and the solution turns turbid as a result of PRE condensation. (c) Normalized AT590 fluorescence intensity at λ_max = 650 nm plotted over pH for the 25 μM AT532-PRE-h-46 + AT612Q-PRE-h-46 mixture in PBS¹⁴⁰. Each data point was dialyzed to the indicated pH (0.2 pH unit steps). Fluorescence peaks were obtained at 15 and 37 °C (after 5 min equilibration). At 15 °C, the PRE-h-46 remains soluble under all pH conditions, and the fluorescence does not switch (Figure 3b). At 37 °C, the condensation occurs at pH_t = 5.2 with a sharpness of approximately 0.3 pH units between the “ON” and “OFF” state.

Figure 5

(a) GDL hydrolyzes into gluconic acid, which leads to slow and steady acidification over time. (b) pH calibration was performed upon addition of 15 mg/mL GDL to PBS¹⁰⁰. The solid black line shows average of three independent calibrations. Red area shows the standard deviation. (c) Time-lapse fluorescence images of AT532-labeled PRE-h-46 at different pH values. At pH 5.97, the first condensates can be observed (a few are indicated by white arrows). Condensates continue to coalesce and grow. Scale bar: 25 μm.

Figure 6

Sequential (mixed) condensate formation at the microscale at room temperature. (a) Mixtures of 25 μM Cy5-PRE-h-36 and 25 μM FITC-PRE-h-41 in PBS¹⁰⁰ acidified with 15 mg/mL of GDL at different time/pH points. From previous turbidity experiments, PRE-h-41 and PRE-h-36 were expected to form condensates at the same pH (see Figure 3b). The pair formed mixed condensates starting at pH 6.21. No clear signs of multiphase condensation were observed in FITC and Cy5 fluorescent channels. Scale bar: 25 μm. (b) Mixtures of 25 μM Cy5-PRE-h-36 and Cy3-PRE-h-58 in PBS¹⁰⁰ acidified with 15 mg/mL GDL at different time/pH points. From previous turbidity experiments Cy5-PRE-h-36 and Cy3-PRE-h-58 were expected to form condensates at the same pH (Figure 3b) and this was indeed the case: Cy5-PRE-h-36 first formed condensates at pH 5.82, followed by Cy3-PRE-h-58 at pH 5.24. Interestingly Cy3-PRE-h-58 appeared to wet the surface of Cy5-PRE-h-36 condensates and ultimately formed mixed condensates. Scale bar: 25 μm. (c) Insert of wetting behavior of Cy3-PRE-h-58 at pH 4.91 in the Cy3 channel. The pixel intensity of the white dashed line was quantified and plotted, showing a clear accumulation of intensity at the edges of the condensate. “Cy5, FITC, Cy5” indicates the fluorescent channel at which data were recorded. Combinations “FITC Cy5” and “Cy3 Cy5” indicate overlaid/merged data.

Figure 7

Sequential (mixed) condensate formation in microcompartments at room temperature. (a) Top: schematic of microfluidic production of double-emulsion droplets with different types of fluorescently labeled PRE polymers. Bottom: acidification is induced by the addition of PBS¹⁰⁰ (pH 2) in the external environment. Acidification occurs slowly over time due to proton flux across the oil membrane. Depending on their pH_t, different ELP species will phase separate at different times (t₁ and t₂), possibly leading to mixed condensates in the end. (b) Fluorescence stills of droplets containing Cy5-PRE-h-36 and Cy3-PRE-h-58 at 25 μM each acidified over time with time points t₀, t₁, t₂, and t_end indicated. Cy5 and Cy3 as well as Cy5 and Cy5 overlapped/merged fluorescent channels are shown. At t_end, a fully mixed condensate is observed. Scale bar: 50 μm.