Dynamic allostery of Sec machinery in protein transport and folding

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The proposal aims to delineate the molecular mechanism of protein translocation by the Sec system. This machinery provides the main pathway for protein secretion and membrane protein insertion across cellular membranes, and is conserved among all forms of life. Transport of proteins across the bacterial inner membrane occurs primarily at the SecYEG translocon. In this case, secretion mostly occurs post-translationally, with the help of cytosolic ATPase SecA, which associates to drive the protein through the SecY-channel, using energy from ATP hydrolysis and the trans-membrane proton motive force (PMF). Structural biology has revealed the arrangements and interactions between SecYEG and SecA, and provides framework for the project. However, despite this detailed information, the central question of how a polypeptide is dynamically translocated through, or into, the membrane remains to be answered.
This proposal builds on our discovery of two-way allosteric communication between the SecA ATP binding site and the central channel of SecY enabled by advances in single molecule detection. We aim to elucidate the mechanism of this dynamic allosteric coupling and the role of pore dynamics during protein translocation for: secretion, as well as for integral membrane protein insertion and outer membrane protein folding and insertion. We will use a combination of biochemistry and time-resolved single molecule fluorescence and computational tools to follow translocation, and corresponding conformational changes. Förster resonance energy transfer (FRET) will allow real time dynamic reporting for interpretation in the context of the available high-resolution structures.
The results will address the key outstanding question: how rapid, stochastic gating of the translocon is allosterically coupled to slower ATP hydrolysis at the SecA motor, and whether such dynamic coupling is required to fulfil additional insertion and downstream functions of this versatile membrane machinery.

The overarching aim of the proposal is to gain an understanding of an important fundamental aspects of bacterial biology: protein secretion, membrane protein insertion and Gram-negative outer-membrane biogenesis. The immediate impact in terms of the current project will lie in scientific advancement and the generation of new knowledge. The project will also present new hypothetical concepts that if proven to be true will have a major impact in our understanding of protein transport, and have important implication for the development of effective treatments against bacterial infections. An additional goal is to encourage a broader uptake of the technological applications we are helping to develop for exploitation in fundamental studies in both academic and commercial sectors.

The main areas of impact are:
1. Application and exploitation. While the proposed project is at a "pre-competitive" stage in terms of commercial exploitation, the knowledge generated will have an immediate benefit to both the National and International bioscience community (academic and commercial) in terms of understanding a fundamental process that spans the breadth of biology. The process is of fundamental importance for bacterial survival and certain complex components are specific to bacteria. The bacterial envelope and its biogenesis are particularly vulnerable to attack; its weakening by, for instance, antibiotics can be lethal. Therefore, the subject of this proposal is a particularly fertile area, with respect to the development of new antibiotics and for strategies against anti-microbial resistance (AMR). Therefore, in the medium term the work could lead to new approaches/ targets for antimicrobial drug development. The knowledge gained could support an ongoing work aimed towards a drug discovery programme in Bristol.

2. Development of new technology. Single molecule detection is emerging as important screening tool as demonstrated in Leeds by the development of sensitive methods to follow virus assembly and screen for anti-virals.

Both Leeds and Bristol have mechanisms in place to increase the impact of research and to exploit any commercialization.

3. Engagement. The benefits to the bioscience community are summarised above. The standard routes to information dissemination (e.g. pre-print submissions, papers in journals and presentations at conferences) will be used throughout the duration of the project. A more general benefit of our work to the UK stems from our commitment to public engagement. The PI and PDRAs routinely participate in public engagement activities, from school children to politicians, and for the promotion science to women and girls. The group will continue with public engagement activities throughout the course of the project, using work generated from the project to exemplify the importance of research.

The PIs also interact with pre-university students with the aim to excite them about the research process in order to encourage them to pursue a future in the high value field of research and development. The PIs will continue with these activities throughout the course of the project, using work generated from the project to exemplify the importance of research.

4. The critical collaboration proposed with the Tuma group in the Czech Republic, which will enhance the value of research in the UK and maintain the UK's scientific European research network, which post-BREXIT will be more important to maintain than ever before.

5. Staff training. The project will generate trained staff with desirable expertise in complex biochemical and biophysical analysis of membrane protein complexes that are involved in important bacterial activities. The researchers will be in demand in both the academic and commercial sectors. During the project, further development will be encouraged through attending courses in areas directly and indirectly related to their role as research scientists (e.g. management and leadership).