Crystallization of a Wild Type Viral Cysteine Protease

Viral proteases have been implicated in harmful viral diseases, and as such are becoming increasingly relevant drug targets. Essential for drug design is a detailed knowledge of the structure of the drug target, particularly its active site and binding regions. Research in this area, however, is lacking and many protease structures remain unknown. This project aims to advance towards solving the structure of a viral cysteine protease through means of crystallization and X-ray crystallography. The wild-type protease was crystallized using sitting drop vapor diffusion. Due to the dynamic nature of proteins, crystallization is no easy feat. Initial high throughput screens using commercial kits streamlined the identification of conditions with potential to form crystals. This was followed by several rounds of optimization of crystallization parameters including pH, buffer concentration, salt concentration, additive type, and crystallization volume in an effort to find a condition suited for crystal growth. This process eventually resulted in large, well-ordered crystals suitable for further experimentation. X-ray crystallography performed on these crystals demonstrated significant diffraction patterns up to a resolution of 3.7 Angstroms, indicative of moderate success in crystallization. Initial data processing was able to identify space groups, but analysis beyond that requires a higher resolution diffraction pattern. Future research will focus on continuing to optimize crystallization conditions to obtain a higher quality crystal and thus higher resolution diffraction patterns. From that point, diffraction data can be processed to build a structural profile of the wild-type viral protease. This information will serve invaluable for future research on the structures of similar proteins and for structure-based drug design targeted towards this viral protease.