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Substrate-transporter complexes in the twin-arginine protein transport system

Reference	BB/F02150X/1
Principal Investigator / Supervisor	Professor Benjamin Berks
Co-Investigators / Co-Supervisors
Institution	University of Oxford
Department	Biochemistry
Funding type	Research
Value (£)	373,829
Status	Completed
Type	Research Grant
Start date	01/01/2009
End date	31/12/2011
Duration	36 months

Abstract

The Tat system of bacteria and chloroplasts carries out the unusual, and mechanistically challenging, task of moving folded proteins across biological membranes. The membrane proteins TatA, TatB and TatC are the essential components of the Tat pathway. During the Tat translocation cycle they are believed to form a series of dynamically changing complexes with the substrate protein. This proposal builds on our success in developing methods to trap, purify in biochemical quantitites, and structurally characterize a transport intermediate involving TatB, TatC, and substrate. A series of experiments will probe Tat mechanism by: [a] Attempting to trap and purify analogues of the fully assembled translocation site containing substrate and TatA, TatB, and TatC. [b] Testing whether the structural re-arrangement of the TatBC complex upon substrate binding that we have identified by electron microscopy (i) requires the transmembrane proton electrochemical gradient (ii) involves protomer loss from the complex. [c] Testing whether the transmembrane proton electrochemical gradient causes the TatBC complex to bind substrate more tightly. [d] Exploiting our discovery of a stable TatAC-substrate complex to (i) investigate the nature of the interactions of TatA with TatC and with substrate and (ii) to investigate the different functional roles of the homologous TatA and TatB proteins.

Summary

Some proteins operate on the outside of the bacterial cell, for example the toxins produced by bacterial pathogens. Since all proteins are made inside the bacterium the extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by machines termed protein transporters that are located in the cell membrane. One type of transporter moves unfolded proteins, threading them across the membrane like string through the eye of a needle. By contrast, a second type of transporter, which we term the Tat system, moves folded proteins across the membrane. This is much more challenging than threading and so it is thought that the Tat system operates by an unusual mechanism. The Tat system is required for many bacterial processes including energy generation, cell division, pathogenesis, and the nitrogen-fixing symbiosis of soil bacteria with plants. The Tat protein transport system is not only found in bacteria but is also present in the chloroplasts of plants where it is essential to form and maintain the proteins required to carry out photosynthesis. The heart of the Tat system is a complex formed by two proteins called TatB and TatC. This complex sits in the cell membrane and recognises and binds the proteins that are to be transported. It then binds another protein called TatA to form the active transporter. This project aims to characterise the complexes formed between the Tat components and the transported protein in more detail with the aim of increasing our understanding of the unusual mechanism of the Tat system. The Tat system is a possible drug target because it is required for bacterial pathogenesis but is not found in humans. It is also of biotechnological interest because it could be utilised to secrete useful protein products.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
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

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