CRYSTALLOGRAPHIC APPROACHES TO OBTAIN STRUCTURAL SOLUTIONS FOR DYNAMIC CAPSID PROTEINS MEDIATING VIRAL ASSEMBLY

The assembly pathway of Bacteriophage λ is conserved in many eukaryotic and prokaryotic viruses. The Major Capsid Protein (gpE) is a subunit of the virus protein head that is assisted by an intrinsically disordered scaffold protein (gpNu3) to polymerize into an icosahedron shell. gpE forms hexamer and pentamer intermediates called capsomers; however, the initial steps of gpE forming these intermediate structures are unclear. Determining an atomic-resolution image via X-ray diffraction (XRD) can reveal details of unique protein-protein interactions that occur when these proteins modulate assembly pathways seen in eukaryotic and prokaryotic viruses. Structural determination of capsid and other viral proteins can serve as targets for drug discovery for treatment of viral infections or development of phage nanotechnologies as therapeutics to target diseases using phage-display conjugated ligand delivery systems. Atomic structural characterization of the gpE-gpNu3 complex (ENu3) is lacking due to the instability of the complex spontaneously self-assembling into the capsid which is influenced by the disordered folding. In this work, novel crystallographic approaches were utilized to improve crystal size and morphology which potentially can solve the ENu3 complex structure. The ENu3 protein complex was co-expressed and purified by anion-exchange chromatography and size-exclusion chromatography. We obtained one fraction that contained pure gpE and gpNu3 with sizes of 38 kDa and 18 kDa, respectively. The proteins in this fraction formed needle clusters during the purification process. Microscale Matrix Seeding (MMS) was applied to further increase the improvement of size of needle clusters. Multiple rounds of MMS yielded novel rectangular plate-like crystals that will be used to collect XRD data and solve the structure. In this report, we discuss our work related to improvements to the protein purification and crystallographic processes as a step towards obtaining the atomic structure of ENu3.