Understanding the Mechanism of Membrane Protein Insertion

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The structural analysis of membrane proteins has heralded an extraordinary enrichment of our understanding of their diverse activities. However, the mechanism governing their insertion into the membrane is poorly understood. This outstanding problem will be addressed through the analysis of the ubiquitous Sec-machinery, responsible for both protein secretion and insertion. The proposal will build on the production of the bacterial holo-translocon (HTL), comprising the SecYEG protein channel complex, the accessory sub-complex SecDF-YajC and the membrane 'insertase' YidC. Their availability has enabled the reconstitution of co-translational membrane protein insertion from pure components and the determination of the structure of the machinery by electron cryo-microscopy. Fitting individual component structures (SecYEG, SecDF-YajC and the periplasmic domain of YidC) to the available EM map suggests the presence of a large partially enclosed cavity that we propose contains lipids. This lipid-pool may provide a protected environment for the insertion of membrane proteins, prior to release into the bilayer.

The hypothesis will be tested through a comprehensive analysis of the activity and structure of HTL. An important first step will utilise the pure reconstituted system in order to describe the basic biochemistry of the system (substrate specificity, bioenergetics, etc). These studies will be enhanced by new synthetic biology methods to expand the capabilities of the classical biochemical and biophysical approaches employed so far. Genetic code reprogramming will be exploited to incorporate non-natural amino acids with unique fluorescence and photo-crosslinking chemistry at defined positions in substrate membrane proteins. The aim is to decipher the environment and pathway of the inserting protein and the corresponding architecture of the machinery. The information will prove decisive for the proposed hypothesis and for understanding the underlying mechanism.

The overarching and immediate aim of the proposal is to gain an 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 protein translocation and potentially the study membrane proteins and protein complexes in general. The project will bestow the benefits of using emerging synthetic biology approaches to address problems of fundamental biological importance. This is exemplified through the use of a reprogrammed genetic code to expand the chemical reactivity sampled by proteins, which will encourage a broader uptake of the approach 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. The process is of fundamental importance for bacterial survival and certain complex components are specific to bacteria. Therefore, in the medium term the work could lead to new approaches/targets for antimicrobial drug development. The knowledge gained could support an on going drug discovery programme (collaboration with Dr A. Woodland, Drug Discovery Unit, Dundee) aimed at the identification of small molecule inhibitors of the bacterial translocon. A second aspect is the generation of bionanodevices through the use of engineered in vitro membrane-protein systems. For example, membrane channels akin to SecYEG are already being exploited in advanced DNA sequencing approaches. Finally, the new synthetic biological approach proposed has implications in terms of its use in other protein complexes. Non-natural amino acid incorporation opens the ability to introduce a wide range of useful chemistry that will greatly facilitate the acquisition of high resolution and value data currently out of reach of existing approaches. Both Bristol and Cardiff have mechanism 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. 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 synthetic biology and complex biophysical/biochemical analysis of 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 generate a new collaboration that brings together groups with different but mutually compatible research areas.