In vitro analysis of Tat protein transport using single molecule fluorescence methods

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 Tat system is a possible drug target because it is required for bacterial pathogenesis but is not found in humans or animals. It is also of biotechnological interest because it could be utilised to secrete useful protein products. This project aims to use cutting edge technology to elucidate major features of how the Tat machinery works. The key to our method is to modify the protein components of the Tat machinery so that they emit light when illuminated by an appropriate light source. We will then used advanced microscopy to visualise individual Tat proteins as they carry out transport. We hope to be able to watch how the different proteins come together and then apart again during the transport process and to follow the transported protein as it travels across a membrane. Analysis of how these events occur, and how much time it takes to complete each step, will significantly advance our understanding of the Tat transport process. This knowledge will help underpin the exploitation of the Tat pathway for useful purposes.

The Tat system of bacteria and chloroplasts carries out the unusual, and mechanistically challenging, task of moving folded proteins across biological membranes. The Tat transport cycle is driven by the transmembrane proton electrochemical gradient and appears to involve dynamic changes in the polypeptide composition of the Tat translocation apparatus. However, the mechanism of Tat transport remains to be elucidated. We have recently been successful in characterizing Tat complexes in living cells using single molecule fluorescence techniques and in establishing a fluorescence-based in vitro Tat transport assay. We now seek to build on this work by applying single molecule fluorescence methods to study Tat transport in a well-controlled in vitro system based on the integration of native bacterial membrane vesicles into a droplet/hydrogel bilayer. Specifically we aim to: [a] Determine the subunit composition of the Tat complexes formed at different stages of the Tat transport cycle and follow the kinetics of the interconversion between these states. [b] Directly test the proposal that substrate-induced polymerization of the TatA component occurs during Tat transport. [c] Characterize the binding of substrate molecules to Tat components and solve a controversy over the number of functional substrate binding sites in the TatBC complex. [d] Obtain single molecule kinetic date for the individual steps in substrate transport. [e] Determine which steps in the transport mechanism require energization by the protonmotive force.

This is hypothesis driven research. However, our results will be relevant in underpinning commercial efforts to exploit the Tat pathway - for production of proteins of therapeutic and industrial relevance - as an analytical tool for quality control of protein folding - as a target for novel antimicrobials The novel, patented droplet/hydrogel interface bilayer technology used in this study is under development for drug screening and other applications. The results of this study will expand the knowledge base concerning this novel technology and its potential. Communication with potential industrial beneficiaries will take place via the technology transfer infrastructure of the University of Oxford. Specifically, we will patent intellectual property arising from this research, and then seek to license or spin-out this technology with the support of Isis Innovation Ltd. Co-investigator Wallace has experience of this process. The primary mechanism for communication of this research will be through publication in peer review international journals. Open access publishing options will be used where available. We will liaise at the time of publication with the University of Oxford and BBSRC Press offices to ensure publicity of results of interest to the general public. Our results will also be made available on our regularly updated web sites. The researchers employed on this grant will gain technical skills in cutting edge bionanotechnology and quantitative data analysis as well as writing, IT, and presentational skills.