Cromoglycate mesogen forms isodesmic assemblies promoted by peptides and induces aggregation of a range of proteins

Abstract

Disodium cromoglycate (5'DSCG) belongs to a class of nonamphiphilic molecules that form nematic chromonic liquid crystals in aqueous solutions. As the concentration increases, it is believed that the molecules first form isodesmic assemblies in water, which further align to form liquid crystal phases. However, the reports on isodesmic assemblies of 5'DSCG have been scarce. Herein, we show that the presence of peptides can promote the isodesmic assembly of 5'DSCG over a broad range of concentrations before reaching the liquid crystal phase. The presence of peptides can lower the 5'DSCG concentration in the aqueous solution to ∼1.5 wt% (from 11-12 wt%, forming a nematic liquid crystal phase) for isodesmic assembly formation. This result indicates a demixing between 5'DSCG and peptides in aqueous solution. We further explored this demixing mechanism to precipitate a wide range of proteins, namely, lectin A, esterase, lipase, bovine serum albumin, trypsin, and a pilin protein from bacterium Pseudomonas aeruginosa. We found that 5'DSCG caused the aggregation of all these proteins except trypsin. These results, along with past findings, suggest that 5'DSCG isodesmic assemblies have the potential to assist in protein purification and crystallization.

Fig. 1. Schematic of the formation of an isodesmic assembly of 5′DSCG, induced by chemical additives.

Fig. 2. Optical density (OD₆₀₀) measurements versus the concentrations of 5′DSCG in deionized water and in LB medium. Arrows indicate the concentrations at which the liquid crystal (LC) phase forms.

Fig. 3. ¹H NMR spectra of 1.5 wt% 5′DSCG without (A) and with (B) LB components, 2.5 wt% 5′DSCG without (C) and with (D) LB components, and (E) LB components in D₂O. The protons of 5′DSCG and peak assignments are labelled in red.

Fig. 4. NOESY spectra of 2 wt% 5′DSCG with (A) and without (B) LB components in D₂O. With LB, cross peaks 1, 2, and 5 are intermolecular NOEs, and 3 and 4 are a mixture of inter- and intramolecular NOEs. Without LB, cross peaks 3 and 4 are intramolecular NOEs.

Fig. 5. NOESY spectra of 2 wt% 5′DSCG with LB components at different temperatures: 21 °C (A), 27 °C (B), 29 °C (C), 31 °C (D), and 33 °C (E). Plot of intensity of NOE cross peak 2 vs. temperature (F).

Fig. 6. Optical density (OD₆₀₀) measurements of the solutions containing 1 wt% peptide mixtures (peptone, casitone, and tryptone) mixed with different concentrations of 5′DSCG. The arrow indicates 5′DSCG concentration at which the liquid crystal (LC) droplets appear.

Fig. 7. Optical density (OD₆₀₀) measurements of solutions containing 3 wt% 5′DSCG mixed with different concentrations of peptides: casitone, peptone, and tryptone.

Fig. 8. Optical density (OD₆₀₀) measurements of solutions containing 7 wt% 5′DSCG mixed with different concentrations of non-ionic polymers: polyvinylalcohol (PVA, M_w ∼ 9000–10 000), poly-vinylpyrrolidone (PVP, M_w ∼ 40 000), and poly-acrylamide (PAAm, M_w ∼ 9000–10 000). Arrows indicate the polymer concentration mixed with 7 wt% 5′DSCG at which the liquid crystal (LC) droplets appear.

Fig. 9. Pilin aggregates induced by different precipitants in hanging droplets. The droplets (5 μL) contained (A) 0.15 wt% 5′DSCG and 50 mg mL⁻¹ pilin, (B) 0.375 wt% 5′DSCG and 5 mg mL⁻¹ pilin, (C) 0.625 wt% 5′DSCG and 25 mg mL⁻¹ pilin, (D) 0.5 wt% NaCl and 10 mg mL⁻¹ pilin, and (E) 1.5 wt% PEG8000 and 5 mg mL⁻¹ pilin. The reservoir solution contained 350 μL of (A) 0.30 wt% 5′DSCG, (B) 0.75 wt% 5′DSCG, (C) 1.25 wt% 5′DSCG, (D) 1 wt% NaCl, and (E) 3 wt% PEG8000. Hanging drops kept at ambient temperature were observed over 5–15 days. All solutions were prepared using 25 mM Tris buffer, pH = 7.5. Scale bar = 380 μm.

Fig. 10. Truncated pilin aggregates induced by different precipitants in hanging droplet. The droplets (5 μL) contained 0.375 wt% 5′DSCG & 5 mg mL⁻¹ truncated pilin, without polarizer (A), and under cross polars (B); 0.625 wt% 5′DSCG & 1 mg mL⁻¹ truncated pilin, without polarizer (C), and under cross polars (D); 1.25 wt% 5′DSCG & 5 mg mL⁻¹ truncated pilin, without polarizer (E), and under cross polars (F); 0.15 wt% 5′DSCG & 0.375 wt% NaCl & 2.5 mg mL⁻¹ truncated pilin without polarizer (G) and under cross polars (H) and 25 wt% PEG4000 & 1 mg mL⁻¹ truncated pilin without polarizer (I). The reservoir solution contained 350 μL of (A and B) 0.75 wt% 5′DSCG, (C and D) 1.25 wt% 5′DSCG, (E and F) 2.5 wt% 5′DSCG and (G and H) 0.3 wt% 5′DSCG & 0.75 wt% NaCl, and (I) 50 wt% PEG4000. Hanging drops kept at ambient temperature were observed over 5–15 days. All solutions were prepared using 25 mM Tris buffer, pH = 7.5. Scale bar = 76 μm.