Award details

Unravelling the molecular basis of subunit specificity in bacterial pilus assembly mechanisms

Reference	BB/F012128/1
Principal Investigator / Supervisor	Professor Gabriel Waksman
Co-Investigators / Co-Supervisors	Professor Alison Ashcroft, Professor Sheena Radford
Institution	Birkbeck College
Department	Biological Sciences
Funding type	Research
Value (£)	397,455
Status	Completed
Type	Research Grant
Start date	01/07/2008
End date	30/06/2011
Duration	36 months

Abstract

Many pathogenic Gram-negative bacteria, including E. coli and Salmonella enterica, produce proteinaceous fibres, known as adhesive pili, on their surfaces that initiate the invasion of the host and are crucial for pathogenesis. These highly ordered protein assemblies are fascinating biopolymers, being comprised of hundreds of contiguous copies of immunoglobulin domains, assembled non-covalently in a precise order. Each pilus subunit folds, in a chaperone-dependent manner, to a structure based on an incomplete immunoglobulin fold, which is subsequently completed by the donation intermolecularly of a single ?/strand from an adjacent subunit formed by its N-terminal extension (Nte). Despite the importance of this biological self-assembly process from both fundamental and applied viewpoints, the structural molecular mechanism by which proteins assemble into pili is not understood. Key questions concern how the precise order of subunits within the pilus is determined and the role of the chaperone and membrane-bound usher in controlling or adapting the assembly mechanism in vivo. Here we propose to use non-covalent mass spectrometry combined with structural analysis, kinetic and thermodynamic measurements, protein engineering and peptide design, to address these questions. Specifically, we will focus on P pili of uropathogenic E. coli associated with pyelonephritis (Pap) pili as a model, a system we have shown to be highly tractable for the experiments proposed, to determine the roles of the N-terminal extension (Nte), the pilin:chaperone complex and the soluble N-terminal domain of the usher protein and in defining and controlling the order of subunit-assembly. Finally, using our ability to purify intact functional usher protein we aim to develop an assay capable of providing the first insights into pilus assembly at a membrane surface in vitro.

Summary

This is a joint grant application PI: Professor Sheena Radford Co-PIs: Dr. Alison Ashcroft and Professor Gabriel Waksman Reference: G52950X Objectives, Summaries, Technical Summary, Beneficiaries, and Case for Support for this application can be found under Professor Sheena Radford's submission

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/F012284/1 Unravelling the molecular basis of subunit specificity in bacterial pilus assembly mechanisms

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