| Summary: | Gram-negative bacteria assemble multi-subunit pili on their surfaces to aid in the establishment of infection. One such class of pili are assembled by the chaperone-usher pathway. In the periplasm, pilus subunits are bound to a chaperone protein, which donates a l3-strand to complete the subunit's, fold. At the outer membrane, the chaperone dissociates and subunits polymerise, each donating an Nterminal extension (Nte) to complete the fold of an adjacent subunit, in a mechanism named donor-strand exchange (DSE). Pili can be comprised of a single subunit type, or several different subunits, assembled in a specific order which is important for functionality of the pilus. Here, DSE is investigated in molecular detail, to determine the precise. mechanism by which it occurs and to analyse the formation of subunit-subunit interactions and the molecular basis for ordered subunit assembly. In order to achieve this, a robust and accurate mass spectrometric method has been developed, to allow reactions between non-covalent protein complexes to be monitored, and used in conjunction with mutational and computational studies. The identification for the' first time of a transient intermediate in DSE, for the Saf pili from Salmonella enterica, showed the reaction to occur via a concerted mechanism. The first interaction to form between an Nte and a subunit was identified, the 'P5' binding site, and shown to be essential for efficient DSE. An interaction stabilising the DSE product was also identified, and evidence was obtained for a 'zip-in-zip-out' mechanism of donor-strand swap. In the multi-subunit P-pilus system from Escherichia coli, analysis of all 30 pairs of subunits showed that those known to interact in vivo, 'cognate' pairs, consistently underwent DSE in vitro more rapidly than their non-cognate counterparts. The basis for this specificity was discovered to be due to complementarity between the two molecules at the initial sit~ of Nte docking. Furthermore, the accessibility of this site was determined to be responsible for the rate at which a subunit can bind an Nte. The relationship between inherent biophysical properties and other in vivo factors was explored in the context of correctly ordered pilus assembly. In conclusion, detailed molecular insights into DSE in pilus assembly have been attained. This understanding of the molecular recognition, strand-swap mechanism and complex stabilisation involved, in addition to the structurally-encoded specificity of subunit interactions, has greatly enhanced ou'r knowledge of the chaperone-usher pathway at a fundamental level. This study should therefore act as an effective basis to advance considerably future important investigations.
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