Noncovalent polymerization of mesogens crystallizes lysozyme: correlation between nonamphiphilic lyotropic liquid crystal phase and protein crystal formation

Abstract

Crystallization of proteins is important for fundamental studies and biopharmaceutical development but remains largely an empirical science. Here, we report the use of organic salts that can form a class of unusual nonamphiphilic lyotropic liquid crystals to crystallize the protein lysozyme. Certain nonamphiphilic organic molecules with fused aromatic rings and two charges can assemble into stable thread-like noncovalent polymers that may further form liquid crystal phases in water, traditionally termed chromonic liquid crystals. Using five of these mesogenic molecules as additives to induce protein crystallization, we discover that molecules that can form liquid crystal phases in water are highly effective at inducing the crystal formation of lysozyme, even at concentrations significantly lower than that required for forming liquid crystal phases. This result reveals an example of inducing protein crystallization by the molecular assembly of the additives, and is consistent with a new mechanism by which the strong hydration of an assembly process provides a gradual means to compete for the water molecules to enable solvated proteins to form crystals.

Figure 1

(A) Molecular structures of additives. Molecule 5′DSCG-diviol consists of a mixture of 48 mole % meso compound and 52 mole % racemic mixture. (B) Schematic representation of the thread assembly formed by 5′DSCG molecules in water.

Figure 2

Lysozyme crystals induced by 5′DSCG additives in hanging droplets. The droplets (5 μL) contained 37.5 mg/mL of lysozyme with (A) ~ 2.74 mM 5′DSCG, (B) ~2.74 mM 5′DSCG and ~128 mM NaCl, and (C) ~1.37 mM 5′DSCG and ~128 mM NaCl. The reservoir solution contained 350 μL of (A) ~ 5.5 mM 5′DSCG, (B) ~ 5.5 mM 5′DSCG and ~ 256 mM NaCl, and (C) ~ 2 M (NH₄)₂SO₄. Hanging drops kept at ambient temperature were observed over 3–5 days. All solutions were prepared using 50 mM sodium acetate buffer (pH 4.6). Scale bar = 76 um.

Figure 3

Crystallization of lysozyme using 5′DSCG-diviol. The hanging drops (5 μL) contained 37.5 mg/mL of lysozyme with (A) 0.14 wt% (~2.73 mM), or (B) 0.068 wt % (~1.37 mM) of 5′DSCG-diviol in 50 mM sodium acetate buffer (pH 4.59). Reservoir solutions (350 μL) contained the same additives with twice the concentration as that in the respective drops. The drops were kept at 25°C for about 3 to 5 days. Scale bar = 76 um.