Deciphering the allosteric mechanism of protein translocation through membranes

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Our aim is to delineate the molecular mechanism of protein secretion and membrane insertion. The major route for the passage of proteins across and into cellular membranes is via the Sec translocon, which is conserved among all forms of life. In bacteria, the process occurs at the plasma membrane through the SecYEG complex. Secretion is generally a post-translational reaction driven by the cytosolic ATPase SecA, whereas membrane proteins are threaded into the membrane co-translationally. Structural biology has revealed the arrangements and interactions between SecYEG and SecA and provides a framework for the project. In spite of this, the central question concerning how a polypeptide translocation occurs is not known.

This proposal builds on our recent discovery of a two-way allosteric communication between the SecA nucleotide binding site and the channel within SecYEG, and aims at elucidating key mechanistic aspects of polypeptide transport. We will do so by the exploitation of a powerful combination of classical biochemistry, synthetic biology, single molecule fluorescence and molecular dynamics (MD) simulations. The former will allow us to lock the machinery and substrate proteins in defined conformations and simultaneously label them selectively with fluorescent probes. Single molecule Förster resonance energy transfer (FRET) imaging of these samples will directly visualise the conformational changes through the translocon during active transport and address the key question on the mechanism: is translocation mediated by series of stochastic movements or power strokes? Finally, the MD simulations will aid our interpretation of these conformational changes at atomic detail.

The ultimate objective will be the discovery of the underlying molecular mechanism of protein secretion and membrane protein insertion, and the exploitation of these findings in biotechnology in the spirit of Synthetic Biology, and in medicine toward the development of new antibiotics.

The overarching and immediate aim of the proposal is the understanding of an important fundamental biological mechanism: protein translocation across membranes. The immediate impact will lie in scientific advancement and the generation of new knowledge. We will also present a new technological route to understanding membrane proteins in general. This in turn will bestow the benefits of using emerging synthetic biology together with single molecule detection to address problems of fundamental biological importance. This is exemplified by the use of a reprogrammed genetic code to expand the chemical reactivity sampled by proteins, encouraging a broader uptake for technological applications as well as 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. Since the process is essential for bacteria survival the work could open to new targets for antimicrobial drugs and support our ongoing drug discovery programme (collaboration with Dr A. Woodland, Drug Discovery Unit, Dundee). A second aspect is the generation of bionanodevices through the use of engineered in vitro membrane-protein systems akin the membrane channels currently used for DNA sequencing. Finally, the new synthetic biological approach proposed has implications in terms of its use in other membrane protein complexes, including diverse protein translocons and their polypeptide substrates. 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. The outcomes will open avenues for screening anti-bacterial agents and may find broader application to membrane proteins, many of which are drug targets. Both Bristol and Leeds have mechanisms in place to increase the impact of research and to exploit any commercialisation (see main impact summary).

2. Engagement. The benefits to the bioscience community are summarised above. The standard routes to information dissemination (e.g. papers in journals and presentations at conferences) will be used throughout the duration of the project. When appropriate, important findings will be communicated to wider audiences via press releases. A more general benefit of our work to the UK stems from our commitment to public engagement. Both the PIs routinely participate in public engagement activities, including with politicians through requested briefing notes and "SET for Science" activities. 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 public engagement activities throughout the course of the project, using work generated from the project to exemplify the importance of research.

3. Staff training. The project will ultimately generate trained staff with desirable expertise in protein biochemistry and biophysics applied to multi-subunit membrane protein complexes. Such a person will be in demand in both the academic and commercial sectors. During the project, staff development in general will be encouraged through attending courses in areas directly and indirectly related to their role as a research scientist (e.g. project management and leadership). Staff will also be encouraged to help with public engagement activities.

4. Collaboration. The project will allow the continuation of a successful collaborative partnership between Leeds and Bristol bringing together groups with different but mutually compatible research areas.