Understanding the Mechanism of Membrane Protein Insertion

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. Cell membranes act as a molecular "skin", keeping the cell's insides in and separating different biochemical reactions. The barrier needs to be breached in a controlled manner to allow transport of nutrients, waste products and for communication with the outside world; this is achieved by a wide range of membrane-inserted proteins. We understand a great deal about the diverse biological functions that membrane proteins bestow, such as transport, respiration, photosynthesis. However, we know very little about how membranes are formed. In particular, the fundamental process through which proteins are inserted into membranes is poorly understood. Our proposal aims to address this outstanding problem. The process is facilitated by a number of different protein translocation systems (or translocons), including the ubiquitous Sec-machinery responsible for both protein secretion and membrane protein insertion. We aim to learn more about how this particular system works by studying an example from the common gut bacterium Escherichia coli. This is much more experimentally tractable than the human counterpart, but nonetheless should tell us a lot about how similar systems work in our own bodies.

A collaborative project between the Collinson (Bristol) and Schaffitzel (Grenoble) Labs has for the first time succeeded in producing and assembling the complete bacterial membrane protein insertion machinery - aka the holo-translocon (HTL), composed of 7 individual subunits. The availability of this active machinery provides a unique opportunity to study the mechanism of membrane protein insertion. The molecular structure of the complex has been investigated, revealing a partially enclosed internal cavity that we have strong reasons to believe is composed of phospholipids. This lipid pool may provide a protected environment into which individual membrane-spanning segments of protein are inserted prior to their folding and release into the bilayer. This is an attractive hypothesis because it mirrors the way soluble (non-membrane) proteins are folded within a water-filled interior of large chaperone complexes.

The proposal aims to build on these exciting developments to characterise the activity of HTL and explore the progression of an inserting membrane protein through the complex. An important first step will be to exploit our ability to reconstitute the insertion process from purified components and conduct a comprehensive analysis of basic biochemical rules and requirements of the machinery. The work will also employ new synthetic biology methods to overcome the limitations of the classical biochemical and biophysical approaches employed so far. Collinson and Jones (Cardiff) will combine forces to apply genetic reprogramming to introduce non-natural amino acids into proteins that allow the introduction of novel properties into target proteins. This technology will provide the tools to report on the environment of a protein during its passage into the membrane, as well as on the corresponding architecture of the HTL. Combined with the structure of the active complex, this information will challenge and develop the hypothesis involving the encapsulated insertion of membrane proteins.

The results of the project will be important because they relate to an essential and fundamental biological concept, which may then lead to new ideas about its disruption for the development of anti-bacterial drugs. Moreover, the ideas and principles implemented and developed will be accessible to the analysis of other complex membrane protein systems.

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. This in turn will bestow the benefits of using emerging synthetic biology approaches to addressing problems of fundamental biological importance. This is exemplified through 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. The process is of fundamental importance for bacteria 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 gaining 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.