STRUCTURE-FUNCTION STUDIES ON VIRAL-HOST INTERACTIONS KEY TO ANIMAL IMMUNITY

<b>Project Methods</b><br> Recombinant protein expression and purification. We have extensive experience in large-scale protein expression and purification, attested by a number of crystal structures determined in our previous and current lab. The detailed procedures have been published (Deng, Ernst et al., 2005, Deng, Lewis et al., 2008, Deng, Schnaufer et al., 2004).Crystallization procedures Step 1. Proteins will be screened in a small subset of 48 to 96 conditions with 200 to 500 nanoliter drops to investigate their overall solubility.Step 2. Trials will be set up using commercially available kits (Emerald Cryo I&II, Wizard I&II, Salt Rx, Pact Screen, Index Screen, Crystal Screen I&II, Crystal Screen Cryo and Peg Ion Screen; a total of about 800 conditions), in some cases supplemented by in-house made random screens.Step 3. Based on the results from the two previous steps, follow-up matrices will be designed.Steps 4 and more. Successive generations of follow-up matrices will be set up to further optimize the size and diffraction quality of protein crystals.Structure determinations We have ample experience with flash freezing of crystals for cryo-protection as evidenced by many structures that we reported.Model building and structure analysis The programs like ARP/wARP (Perrakis, Morris et al., 1999) and RESOLVE (Terwilliger, 2003) will be used for automatic tracing. Intermediate models will be improved using interactive graphics programs such as XTALVIEW (McRee, 1999), O (Jones, Zou et al., 1991), and COOT (Emsley & Cowtan, 2004).Analysis of the structures will be carried out by programs in the lab or on the web such as DALI (Holm & Sander, 1993) for searching related protein structures, GRASP (Nicholls, Sharp et al., 1991) for analyzing electrostatic and other surface characteristics, ACCESS for calculating buried solvent accessible surfaces (Leslie, 1992), LIGPLOT (Wallace, Laskowski et al., 1995) for analyzing ligand-protein interactions.Binding affinity assays BIACORE binding assay To facilitate BIAcore analysis, we have developed a facile procedure for coupling protein to sensor chips (Meng, Leman et al., 2007, Xiang & Moss, 2001)Virtual Docking and IL-18 BioassyWe have described the experimental procedures in details in the earlier publications (Krumm, Meng et al., 2017, Meng et al., 2007, Xiang & Moss, 1999).Structure based mutagenesis: Based on the expected structures, amino acids at the protein interface or drug binding site whose side chains are predicted to contribute to the binding specificity and affinity will be mutated to Ala.