Selective Crystallization of Proteins Using Engineered Nanonucleants

This study reports for the first time a detailed experimental investigation of protein crystallization in engineered nanoconfined spaces with both controlled pore diameters and narrow pore size distributions. We propose a systematic approach for controlling the nucleation and crystallization of biological macromolecules based on a relationship between the protein radius of gyration (R-g) and specific pore diameter. A series of nanonucleants with ordered mesopores having narrow pore size distributions were prepared. The templates were tested for proteins ranging in molecular weight from 14 to 450 kDa. Well-formed protein crystals were obtained on only one of the five presented nanonucleants for all protein cases tested, highlighting the unique template selectivity exhibited by these nucleants. In addition, concanavalin A and catalase were both crystallized at similar to 2 times lower supersaturation levels than previously reported by any known method. Our observations fully support theoretical studies that predict the enhanced thermodynamic stability of proteins in nanoconfined cavities, including specifically the importance of nucleant pore diameter with respect to protein radius of gyration. The nucleants described here could have major industrial applications for downstream separation and purification of biopharmaceuticals, as well as improved opportunities for the crystallization of complex proteins for structural determination.