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 non-amphiphilic lyotropic liquid crystals to crystallize the protein lysozyme. Certain non-amphiphilic 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.

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

“…9E and 10I). These results, together with those from past studies, 10,11 suggest that 5 0 DSCG is a potent agent for inducing protein aggregation, and when combining with other chemicals, has the potential to induce protein crystallization.…”

“…However, the formation of isodesmic assemblies of 5 0 DSCG in the presence of peptides does suggest a demixing process between the two components. In a previous study, 11 we have shown that 5 0 DSCG at a low concentration ($0.14 wt% or $2.74 mM) caused the crystallization of lysozyme. Furthermore, 5 0 DSCG appears to be non-disruptive to protein folding by being able to facilitate the specic recognition and binding between proteins and their antibodies.…”

“…Liquid crystals (LCs) have been explored for biological studies and applications. [1][2][3][4][5][6][7][8][9][10][11] Among the different types of liquid crystals, lyotropic chromonic liquid crystals are different from conventional surfactant-based lyotropic liquid crystals as the former consist of nonamphiphilic molecules assembled in water and the assemblies further align to form LC phases. 1,[12][13][14][15][16][17][18][19][20][21][22][23][24][25] Due to the nonamphiphilic nature of the mesogen, the assembly of the molecules does not proceed with the assembly process of surfactants (conventional lyotropic liquid crystals).…”

“…Because the chromonic mesogen disodium cromoglycate (5 0 DSCG) does not have a well-separated oily and water-soluble region within its structure, this molecule is benign to protein folding and appears to be highly compatible for biological applications and studies. 10,11 The nematic phase of this molecule in water has been used for studying surface-oriented movement of bacteria for potential biosensing applications, [2][3][4][5][6][7] specic antibody-antigen binding, 10 and protein crystallization. 11 Far below the LC phase concentration, at $0.14 wt%, 5 0 DSCG has been demonstrated to promote the crystallization of lysozyme.…”

“…10,11 The nematic phase of this molecule in water has been used for studying surface-oriented movement of bacteria for potential biosensing applications, [2][3][4][5][6][7] specic antibody-antigen binding, 10 and protein crystallization. 11 Far below the LC phase concentration, at $0.14 wt%, 5 0 DSCG has been demonstrated to promote the crystallization of lysozyme. 11 More surprisingly, this liquid crystal appears to promote the binding between antigens and antibodies, 10 while other conventional surfactant-based liquid crystals do not support antigen-antibody binding, presumably due to their ability to denature proteins.…”