Deciphering the allosteric mechanism of protein translocation through membranes

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description This grant is approaching its end. The Bristol strand of the project (2 years) has ended and the Leeds strand has a few months to run. The project has implemented a state-of-the-art analysis by single molecule spectroscopy (with our collaborative partners in Leeds), we have made great progress towards the description of the molecular mechanism of protein translocation.

Our first series of experiments are published in eLife (Fessl et al 2018). Following on from that, a series of additional experiments have been conducted to address the remaining objectives towards the determination of the molecular mechanism of protein translocation (BB/N017307/1). We anticipate that we will exceed the objectives that were originally set out, and have ample new leads to build an excellent proposal for a new and exciting series of experiments. Indeed, we have recently submitted another manuscript for peer review, available here:

Joel Crossley, Matthew A. Watson, Tomas Fessl, Daniel Watkins, Robin A. Corey, William J. Allen, Tara Sabir, Sheena E. Radford, Ian Collinson, Roman Tuma. Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation. bioRxiv 793943; doi: https://doi.org/10.1101/793943. Submitted to JACS.

Thus, we are very pleased with the progress and the likely imminent delivery of further high quality outputs, and follow on funding.

In addition to the core progress outlined above we were also able to expand the scope of the research programme exploring additional aspects bacterial secretion and protein translocation.

This grant contributed to the discovery of specific cardiolipin-SecY interactions required for proton-motive force stimulation of protein secretion. The results were published recently in PNAS (Corey et al 2018)

Furthermore, the work also contributed to a study exploring ATP-induced asymmetric of pre-protein folding as a driver of protein translocation through the Sec machinery, which was published recently in eLife (Corey et al 2019).

The grant has also contributed to the development of a new research tool:
A HIGH-RESOLUTION LUMINESCENT ASSAY FOR RAPID AND CONTINUOUS MONITORING OF PROTEIN TRANSLOCATION ACROSS BIOLOGICAL MEMBRANES
Available here:

Pereira, G.C., Allen, W.J., Watkins, D.W., Buddrus, L., Noone, D., Liu, X., Richardson, A.P., Chacinska, A. and Collinson, I. (2019) A high-resolution luminescent assay for rapid and continuous monitoring of protein translocation across biological membranes. J. Mol. Biol. 43,1689-1699. doi: 10.1016/j.jmb.2019.03.007.
Exploitation Route The work has demonstrated how new state of the art techniques in single molecule fluorescence can be exploited toward the analysis of a complex multi-subunt membrane protein transport complex.

Additionally, the activities developed to monitor the activity of the translocation apparatus also seem to be generally applicable for monitoring protein transport, with respect to location and kinetics (Pereira et al 2019).
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title A HIGH-RESOLUTION LUMINESCENT ASSAY FOR RAPID AND CONTINUOUS MONITORING OF PROTEIN TRANSLOCATION ACROSS BIOLOGICAL MEMBRANES 
Description Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time-resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes, and allows continuous acquisition with a time-resolution in the order of seconds - yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system, and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigour unattainable through classical methods. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact see paper Pereira, G.C., Allen, W.J., Watkins, D.W., Buddrus, L., Noone, D., Liu, X., Richardson, A.P., Chacinska, A. and Collinson, I. (2019) A high-resolution luminescent assay for rapid and continuous monitoring of protein translocation across biological membranes: bioRxiv - https://doi.org/10.1101/456921 In Press at J. Mol. Biol 
URL https://doi.org/10.1101/456921
 
Description Analysis of the Sec machinery by ESR with Dr Janet Lovett 
Organisation University of St Andrews
Country United Kingdom 
Sector Academic/University 
PI Contribution Samples for ESR
Collaborator Contribution ESR time
Impact None yet
Start Year 2011
 
Description Analysis of the mechanism of protein translocation by single molecule fluorescence with Profs Sheena Radford and Roman Tuma 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of expertise and material. Conducting in parallel ensemble analysis of protein transport machinery See joint BBSRC grants: Recently awarded: BB/T006889/1 (joint with BB/T008059/1) BB/N017307/1 (joint with BB/N015126/1) BB/I006737/1 (joint with BB/I008675/1)
Collaborator Contribution Single molecule expertise, experimental set up and data collection
Impact Yes, publications: Joel Crossley, Matthew A. Watson, Tomas Fessl, Daniel Watkins, Robin A. Corey, Tara Sabir, Sheena E. Radford, Ian Collinson, Roman Tuma. Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation. bioRxiv 793943; doi: https://doi.org/10.1101/793943. Submitted to JACS. Fessl T., Watkins D., Oatley P., Allen W.J., Corey R.A., Horne J., Baldwin S.A., Radford S.E., Collinson I. & Tuma R. (2018) Dynamic action of the Sec machinery during initiation, protein translocation and termination. eLife: 10.7554/eLife.35112 Allen, W. J., Corey, R. A., Oatley, P., Sessions, R. B., Baldwin, S. A., Radford, S. E., Tuma, R., and Collinson, I. (2016) Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation. eLife. 10.7554/eLife.15598 Deville, K., Gold, V. A. M., Robson, A., Whitehouse, S., Sessions, R. B., Baldwin, S. A., Radford, S. E., and Collinson, I. (2011) The oligomeric state and arrangement of the active bacterial translocon. J. Biol. Chem. 286, 4659-4669
 
Description Analysis of the mechanism of protein translocation by single molecule fluorescence with Profs Sheena Radford and Roman Tuma 
Organisation University of South Bohemia
Country Czech Republic 
Sector Academic/University 
PI Contribution Provision of expertise and material. Conducting in parallel ensemble analysis of protein transport machinery See joint BBSRC grants: Recently awarded: BB/T006889/1 (joint with BB/T008059/1) BB/N017307/1 (joint with BB/N015126/1) BB/I006737/1 (joint with BB/I008675/1)
Collaborator Contribution Single molecule expertise, experimental set up and data collection
Impact Yes, publications: Joel Crossley, Matthew A. Watson, Tomas Fessl, Daniel Watkins, Robin A. Corey, Tara Sabir, Sheena E. Radford, Ian Collinson, Roman Tuma. Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation. bioRxiv 793943; doi: https://doi.org/10.1101/793943. Submitted to JACS. Fessl T., Watkins D., Oatley P., Allen W.J., Corey R.A., Horne J., Baldwin S.A., Radford S.E., Collinson I. & Tuma R. (2018) Dynamic action of the Sec machinery during initiation, protein translocation and termination. eLife: 10.7554/eLife.35112 Allen, W. J., Corey, R. A., Oatley, P., Sessions, R. B., Baldwin, S. A., Radford, S. E., Tuma, R., and Collinson, I. (2016) Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation. eLife. 10.7554/eLife.15598 Deville, K., Gold, V. A. M., Robson, A., Whitehouse, S., Sessions, R. B., Baldwin, S. A., Radford, S. E., and Collinson, I. (2011) The oligomeric state and arrangement of the active bacterial translocon. J. Biol. Chem. 286, 4659-4669
 
Description Analysis of translocating protein by NMR with Prof John Christodoulou 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Protein samples for analysis by NMR
Collaborator Contribution Expertise in NMR for labelling and recording spectra
Impact No yet
Start Year 2016
 
Description Application of SMALPS for analysis of the bacterial translocation machinery with Prof Tim Dafforn 
Organisation University of Birmingham
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Protein material for analysis
Collaborator Contribution Reagents to extract and analyse the protein material
Impact Komar, J., Alvira, S., Schulze, R., Martin, R., Lycklama a Nijeholt, J., Lee, S., Dafforn, T., Deckers-Hebestreit, G., Berger, I., Schaffitzel, C., and Collinson, I. (2016) Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. Biochem. J. 10.1042/BCJ20160545
Start Year 2015
 
Description Computational analysis of the bacterial translocon with Dr R. Corey 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in empirical analysis of the bacterial secretion machinery
Collaborator Contribution Expertise for computational analysis of the bacterial secretion machinery
Impact None so far
Start Year 2018
 
Description Mass spectrometry of the bacterial Sec machinery with Dr A. Politis 
Organisation King's College London
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We are conducting experiments using mass spectrometry to characterise the properties of the bacterial secretion machinery. We provide the material for analysis.
Collaborator Contribution Mass spec equipment and expertise
Impact In progress
Start Year 2017
 
Description Protein Biophysics of protein transport apparatus with Dr T. Fessl and Prof. R. Tuma 
Organisation University of South Bohemia
Country Czech Republic 
Sector Academic/University 
PI Contribution Provision of samples for biophysical analysis, especially for single molecule applications
Collaborator Contribution Biophysical analysis of protein transport apparatus, including single molecule applications
Impact Joel Crossley, Matthew A. Watson, Tomas Fessl, Daniel Watkins, Robin A. Corey, Tara Sabir, Sheena E. Radford, Ian Collinson, Roman Tuma. Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation. bioRxiv 793943; doi: https://doi.org/10.1101/793943. Submitted to JACS. Corey, R. A., Ahdash, Z., Shah, A., Pyle, E., Allen, W.J., Fessl, T., Lovett, J.E., Politis, A. and Collinson, I. (2019) ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery. eLife: 10.7554/eLife.41803 Fessl T., Watkins D., Oatley P., Allen W.J., Corey R.A., Horne J., Baldwin S.A., Radford S.E., Collinson I. & Tuma R. (2018) Dynamic action of the Sec machinery during initiation, protein translocation and termination. eLife: 10.7554/eLife.35112
Start Year 2018
 
Description The Bacterial Sec Machinery with Dr Andrew Woodland 
Organisation University of Dundee
Department Drug Discovery Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of samples and expertise for measurement of ATP driven protein transport through the bacterial Sec machinery
Collaborator Contribution Expertise for high-throughput analysis and exposure to large small chemical libraries to search fro potent inhibitors (potential anti-bacterial)
Impact Work in progress
Start Year 2012
 
Description Bath Royal Literary and Scientific Institution (led by postdoc Dr Lisa Buddrus) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact In partnership with the Bath Royal Literary and Scientific Institution and with the support of the University of Bristol Public Engagement Office, I have run several hands-on workshops for children aged 11 to 13 around microbiology and biochemistry, introducing biotechnology, antimicrobials and antimicrobial resistance, and various aspects of hygiene and spread of infections. In partnership with the University of Bath (UoB), I also organised a workshop at the UoB Department of Biology and Biochemistry teaching labs, exploring DNA and synthetic biology. I was also involved in the events at We the Curious during World Antibiotic Awareness Week, demystifying appropriate antibiotic use and spread of antibiotic resistance with children of various ages.
Year(s) Of Engagement Activity 2017,2018
 
Description STEMming Girls: Inspiring New Women Generations in STEM (led by postdoc Dr Sara Alvira de Celis) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact see URL below and also here: http://www.bristol.ac.uk/biochemistry/public/news/2018/international-day-of-women-and-girls-in-science.html
Year(s) Of Engagement Activity 2018
URL https://sruk.org.uk/events/stemming-girls-inspiring-new-women-generations-in-stem/