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अमूर्त

Design and Selection of IFI16-PAAD Mutants with Improved dsDNA Destabilization Properties

Desmond Lau, Kush Dalal, Benjamin Hon and Frederic Pio

The PAAD Death Domain is a 6-helix bundle domain that contains a disordered region in helix-3. This domain can fold by a two-state mechanism, has low stability and can undergo different partially folded conformations. To identify specific amino acids responsible for these structural features we focus our study on the PAAD domain of IFI16, a protein sensor that recognizes viral nucleic acids and triggers the (Interleukin-β) inflammatory response in response to infection. IFI16-PAAD was randomly mutated using GFP directed evolution and the resulting mutants with increased secondary structure and stability were identified by circular dichroism and tested for nucleic acid binding. Several IFI16-PAAD-GFP fusion mutants that contained I46N/S substitutions translated into improved secondary structure and stability and a direct correlation was observed between secondary structure/stability enhancement and their ability to destabilize dsDNA. In conclusion, GFP directed evolution can be used to obtain more structured and stable mutants and to probe conformational changes. These data suggest that changes in conformation of the PAAD domain of IFI16 occurs when the viral nucleic acid bind to IFI16 and must be a contributing factor to transduce the inflammatory response during viral infection.