Characterisation of the filamentous bacteriophages end-caps : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand. EMBARGOED until 30 April 2023.

Embargoed until 30 April 2023Ff (f1, fd, and M13) filamentous phage of Escherichia coli have collectively been the workhorse of phage display technology over the past few decades. Their use has expanded in the recent years into nanotechnology, where they serve as filament-like templates (≥ 880 nm x 6 nm) for assembly of nanostructures such as nanowires, nanorings, and more complex assemblies including nano-scale batteries, among others. The filament end-caps are the key to improving phage display applications and design of novel Ff-built nanostructures. Furthermore, proteins pIII and pVI, that form the distal end-cap, are of key importance for understanding the Ff biology. They mediate the Ff assembly-release and entry, two opposing processes that involve, respectively, excision and insertion of the virion out of and into the inner membrane of E. coli. The mechanisms of these two processes are a mystery, and the only path towards understanding is to determine the structures of the pIII-pVI complex. While the atomic resolution structure of the Ff filament shaft made of the major coat protein pVIII has been determined, the structure of the phage end-caps remains unresolved, as they constitute only 2% of the virion mass. To enable the end-cap structural analyses we developed methodology for high-efficiency production and purification of short Ff-derived nanorods, where the end-caps are highly enriched, accounting for up to 38% of the total virion mass. Furthermore, methods for Ff-derived nanorod production have been majorly improved in this thesis by engineering a novel system, resulting in at least 200-fold increase relative to the systems described previously. The nanorod purification was also improved by including an anion exchange chromatography step. Highly pure and concentrated 50 nm and 80 nm nanorods were analysed structurally and biochemically to characterise the pIII-pVI complex. Intact nanorods were structurally characterised by cryo-EM single-particle analysis (cryo-EM SPA) that resulted in 2D classes of the filament end-caps. Furthermore, a preliminary 3D model of the pIII-pVI cap was generated at a resolution of 5 Å. Further refinement of the 3D model is under way. Besides the intact particles, analysis was expanded to purified pIII-pVI complex obtained from the DOC-chloroform-disassembled nanorods by size exclusion chromatography. Under native conditions, protein pIII could be detected in a complex larger than 720 kDa, indicating that multiple copies of pVI and pIII form a multimer-dimer that includes a substantial amount of the shaft protein, pVIII. Applications of nanorods benefit from precise control of the nanorod lengths, which is very difficult to achieve when it comes to non-biological materials. In this thesis, Ff-derived nanorods of novel sizes were designed by eliminating specific DNA regions from the nanorod replication cassettes that controls the length of the nanorod ssDNA backbone. This work showed that the DNA segment between the packaging signal and (-) ori is not essential for replication and resulted in production of 40 nm nanorods, shortest ever constructed to date. Two novel lengths, 40 and 70 nm, were added to the of sub-100-nm nanorod collection produced using this system