Deciphering the allosteric mechanism of protein translocation through membranes

All cells are surrounded by membranes that act as an ideal "skin", keeping the cell's insides in! In the absence of other components they would act as barriers, preventing the necessary rapid exchange of nutrients and waste products, and of larger molecules like proteins, between the environment and the cell interior. Such passage is required for many proteins to perform their biological functions. To overcome this problem, biological membranes contain a number of translocation systems that enable proteins and other useful substances ("substrates") to pass across and into the phospholipid barrier. In the case of protein substrates, these translocation systems recognise the specific proteins to be translocated via signals embedded in the sequence of amino acids from which they are constructed.

We aim to learn more about how such translocation systems work by studying an example from the common bacterium Escherichia coli, which is experimentally easier to work with than human cells, but nonetheless should inform us how similar systems work in our own bodies. The bacterial translocation system (the "translocon") serves to secrete proteins from the interior of the cell to the outside and to the interior of the membrane itself. It comprises two components - a protein channel through and into the membrane, and a motor protein named SecA that drives the passage of proteins through the channel, fuelled by energy provided by ATP, the so-called "energy currency" of the cell.

We know that the energy for protein translocation is released when SecA breaks down ATP into two smaller molecules, ADP and phosphate. We have a new hypothesis about how this process is coupled to the movement of the translocating protein. It is clear that a cycle of changes in the shapes of SecA and the channel, termed conformational changes, are involved. It is the exact nature of these conformational changes and their precise timescale that will be explored in the proposed project. To do this, we will use genetic and biochemical techniques to introduce optical reporters on the protein substrate and at places in the translocon that we suspect move during the translocation process. The selective attachment of fluorescent probes will report on the environment at each place during different stages of protein translocation and during ATP breakdown.

These types of experiments, conducted in the test tube on millions of translocons at a time under so-called 'ensemble' conditions have been very revealing. However, in such ensembles it is very difficult to synchronise the translocation 'machines' so that they are all simultaneously at the same stage of their mechanical cycles when we observe them. To complement this approach we will therefore also take advantage of the development of very sensitive microscopy techniques, which will allow us to follow the conformational changes of a single translocon, and the associated translocation of protein, at a time. Taken together, the ensemble and single molecule approaches should allow us to test our new model describing how protein translocation works. For instance, they will allow us to distinguish between a 'power-stroke' processive mechanism or a 'rachetting' stochastic one that biases the diffusion of the translocation substrate in the desired direction.

New information on the general basis and finer details of protein seretion and membrane protein insertion that this work uncovers will further our understanding of a fundamental process in biology, occuring in every cell in every organism. Moreover, the findings could be exploited in the development of useful tools inspired by biology, within the burgeoning sphere of Synthetic Biology. In addition, as the project will focus the essential bacterial secretion machinery, the results may aid in the development of compounds that target the elements that are unique to bacteria, and thus with potential highly desirable novel anti-bacterial activity.

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.