Turning Down the Inhibition Effect of Silica Gels in Protein Crystallization

Silica gels act as nucleation inhibitors and have been used to grow large protein crystals in convection-free environments. However, a large amount of protein is required to overcome the inhibition effect, and chances of successful crystallization are limited, hampering its potential benefits. In the present study, we propose the substitution of silanol groups with methylated additives to increase the hydrophobicity of the gel network, decrease the interaction between proteins and gel fibers, and tune the inhibition effect of silica gels. We observed an increased hen egg white lysozyme (HEWL) nucleation density in gels bearing a higher number of methyl groups. We used the counter-diffusion crystallization technique for our proof of concept since it does not require a fine adjustment of the supersaturation. We then moved to batch crystallization for maintaining constant supersaturation conditions in order to have comparative results. We were able to grow HEWL crystals with tailored sizes depending on the amount of hydrophobic moieties' substitution. The modification of the gel reduced the amount of protein required to induce nucleation. This effect was attributed to the decreased adsorption of protein macromolecules on gel fibers carrying hydrophobic groups. This simple chemical modification approach may expand the use of silica gels, traditionally seen as protein nucleation inhibitors, to produce new crystalline composite materials.