Modification of protein crystal packing by systematic mutations of surface residues: implications on biotemplating and crystal porosity.

Bioinspired nano-scale biotemplating for the development of novel composite materials has recently culminated in several demonstrations of nano-structured hybrid materials. Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein "building blocks" in the crystal, a complementary 3D array of interconnected cavities--voids array, exhibiting highly ordered porosity is formed. The porous arrays of protein crystal may serve as a nano-structured, accurate biotemplate by a "filling" process. These cavities arrays are shaped by the mode of protein packing throughout the crystallization process. Here we propose and demonstrate feasibility of targeting site specific mutations to modify protein's surface to affect protein crystal packing, enabling the generation of a series of protein crystal "biotemplates" all originating from same parent protein. The selection of these modification sites was based on in silico analysis of protein-protein interface contact areas in the parent crystal. The model protein selected for this study was the N-terminal type II cohesin from the cellulosomal scaffold in ScaB subunit of Acetivibrio cellulolyticus and mutations were focused on lysine residues involved in protein packing as prime target. The impact of systematically mutating these lysine residues on protein packing and its resulting interconnected cavities array were found to be most significant when surface lysine residues were substituted to tryptophan residues. Our results demonstrate the feasibility of using pre-designed site directed mutations for the generation of a series of protein crystal biotemplates from a "parent" protein.