Ensemble and single molecule analysis of protein translocation

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. These 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 - for example the abundant protein albumin of the blood has to be secreted across the membrane from its site of synthesis in liver cells. To overcome this potential problem, biological membranes contain a number of translocation systems that enable proteins and other useful substances ('substrates') to pass across 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 gut bacterium Escherichia coli, which is experimentally easier to work with than human cells, but nonetheless should tell us a lot about how similar systems work in our own bodies. Like our own, the bacterial translocation system (the 'translocon') serves to secrete proteins from the interior of the cell to the outside. It comprises two components - a three-protein complex named SecYEG that forms a channel through 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 the motor protein SecA breaks down ATP into two smaller molecules, ADP and phosphate. What we don't understand is how this process actually drives movement of the translocating protein. However, it is clear that a cycle of changes in the shapes of SecA and SecYEG, termed conformational changes, are likely to be involved, much as the movements of pistons and cams are involved in internal combustion engines. It is these conformational changes that will be explored in the proposed project. To do this, we will use recombinant DNA techniques to introduce the amino acid cysteine into the protein substrate and at places in the translocon that we suspect move during the translocation process. This particular type of amino acid is chemically reactive, meaning that we can selectively attach fluorescent or magnetic probes with which we can monitor the environment at each place during different stages of protein translocation and ATP breakdown. In particular, the distances between pairs of probes can be measured by physical techniques known as Förster resonance energy transfer (FRET) and electron spin resonance (ESR) respectively. We will also examine whether pairs of cysteines are sufficiently close to each other to be chemically linked together by cross-linking molecules of defined length, and if so, we will see what effect this tethering together has on the function of the translocation machinery. These types of experiments, conducted in the test tube on millions of translocons at a time under so-called 'ensemble' conditions, should be very revealing of the mechanism. 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 understand the inner workings of a molecular machine essential in all cells.

Technical Summary

Protein secretion in bacteria utilises SecA to drive protein through the ubiquitous SecYEG complex. In spite of our knowledge of the structure, and of the stages and timing of the ATP hydrolytic cycle, we understand little about the corresponding conformational changes. We therefore propose a multi-disciplinary programme to explore the dynamics of the translocation machinery. To this end, cysteines will be incorporated to enable selective modification at specific sites in SecA, SecYEG and pre-protein substrate. Intra- and inter-molecular cross-links between specific thiol pairs in SecA and SecYEG will be used to monitor their relative locations in the presence of ADP, ATP (AMPPNP), and when engaged in translocation. Fully cross-linked samples will also be characterised with respect to ATP hydrolysis and pre-protein transport. In addition, the introduction of single or pairs of fluorescent or paramagnetic probes will be used to report on their environment and spatial relationships (e.g. distance and orientation). FRET will be used to monitor nucleotide- and pre-protein dependent conformational changes within SecA and SecYEG. In parallel, ESR spectroscopy will be employed to provide reliable distance constraints between given points of the complex at different stages of the translocation cycle. Ensemble experiments will be complemented by investigations at the single molecule level, using total internal reflection fluorescence microscopy (TIRF). These will allow us to circumvent problems associated with inherently inefficient ensemble assays of transport, and the difficulty of synchronising populations of translocating complexes. It should thereby be possible to follow translocation in real time, including conformational changes in SecYEG and SecA. Taken together, these approaches should contribute greatly to an understanding of the molecular mechanism of protein translocation, a process of critical importance to all cells.

Planned Impact

The main objective of the proposal is toward the understanding of an essential and ubiquitous reaction in cells: protein translocation across membranes. Therefore, the immediate impacts will be in the scientific advancement in the areas of membrane biology, molecular motors, protein dynamics as well as the development of novel biophysical techniques to observe such systems. In addition, it will also offer training in multi-disciplinary research to its post-doctoral workers, which will equip them with new skills and give them essential experience for research or related jobs in academia, education, healthcare, or industry. In addition to the project itself, we anticipate considerable added benefit and impact through collaborative links already established nationally and internationally, particularly amongst other academic institutions in addition to Bristol, Leeds and Oxford. New findings will be disseminated both through peer-reviewed publications and by presentations at international scientific meetings. The output resulting from this project will also be relayed to the media via press release and high profile sites on the Universities' front web pages. Moreover, we will continue our commitment and track-record in public engagement. This involves communication of the excitement of science to the both junior and adult members of our society. The immediate beneficiaries of this project will clearly be the wider UK and international academic communities, public and private education, the healthcare sector and industry. However, in the medium term we foresee potential impacts in the areas of antimicrobial drugs and in nanotechnology. The former reflects the fact that while the translocon per se is ubiquitous, the motor protein SecA, is found only in bacteria. Given the growing problem of antibiotic resistance in the UK and worldwide, such development would have a huge impact on human and animal health. Our findings on the conformations and dynamics of this protein will open the way to the development of novel antimicrobial drugs by the pharmaceutical industry. And in this endeavour we propose to act positively. Should the grant be funded we will set in motion the initial applications for seed-funding and the installation of experimental approaches to inhibitor design of this bacteria specific process. In doing so we will enlist the existing entrepreneurial infra-structures in Bristol and Leeds. The second potential biotechnological application relates to the aspect of the proposal that seeks the understanding of molecular machines utilising chemical energy for molecular motion of proteins across the barrier of the membrane. Activities such as these are of course potentially extremely useful to us. Moreover, our greater understanding of them, particularly in the dynamic energy coupling steps, will enhance our ability to manipulate them accordingly. This could indeed prove useful in the development of novel activities pertinent to bio-sensation or the construction of nano-machines or motors, relevant to the BBSRCs commitment to synthetic biology. Therefore, as the opportunity arises these anticipated spin-offs will be exploited to the full.

Publications

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Corey RA (2016) Protein translocation: what's the problem? in Biochemical Society transactions

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Berger I (2017) Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon. in Methods in molecular biology (Clifton, N.J.)

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Collinson I (2015) Channel crossing: how are proteins shipped across the bacterial plasma membrane? in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Schulze RJ (2014) Membrane protein insertion and proton-motive-force-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We are investigating the mechanism cells use to secrete (export) proteins and insert them into the membrane. We do so by studying the genetically tractable bacteria E. coli. The machinery we study - the Sec machinery - is found in every cell in every organism, so what we learn about the bacterial system will inform how the process works in humans.

Our published findings so far (Gold et al, BJ 2012 and Whitehouse et al, JCB 2012) have helped address the dynamic mechanism of protein translocation across the membrane by examining how specific elements move during protein secretion. Additional, published work (Schulze et al, PNAS 2014) and ongoing work explores how this process occurs during membrane protein insertion, and how it differs from the mechanism of secretion.

This grant has now ended with several publication outputs - see below.

1) We have deciphered the conformational changes induced within the protein-channel (SecY-complex) by the coordinated action of the signal sequence and the motor ATPase SecA. These conformational changes bring about complex dislocation, which enables the intercalation of the translocating polypeptide. The paper describing these results have recently been accepted for publication (Corey et al, Structure 2016).

2) The drive for post-translational transport comes from ATP hydrolysis by SecA, in concert with the trans-membrane proton motive force (PMF). However, the mechanism through which hydrolysis is coupled to force generation is unclear. Using all-atom molecular dynamics simulations of the SecYEG-SecA complex, together with ensemble and single molecule fluorescence resonance energy transfer (FRET), we show that ATP turnover is coupled at long range to opening and closure of the SecY protein-channel. Furthermore, we demonstrate that perturbations within SecY feed back to the ATP binding pocket in SecA to regulate nucleotide exchange. The results are consistent with a Brownian motor mechanism; whereby the opening and closing of the channel are synchronised to the hydrolytic cycle of ATP, co-ordinated also with the translocating pre-protein, to set up a ratchet allowing diffusion across the membrane while preventing backsliding.

This work has now been published in eLife (Allen et al. 2016).

A follow up paper in collaboration with Leeds (Tuma and Radford groups) has also been published on the dynamic action of the Sec machinery during initiation, protein translocation and termination (Fessl et al. 2018).

This work continues through the current award: Deciphering the allosteric mechanism of protein translocation through membranes (BB/N015126/1)

3) Additional work partly supported by this grant has resulted in the analysis of the structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon: see
Martin, R., Larsen, A.H., Corey, R.A., Midtgaard, S.R., Frielinghaus, H., Schaffitzel, C., Arleth, A. and Collinson, I. (2018) Structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon: under revision Biophysical Journal - bioRxiv - https://doi.org/10.1101/490250

4) This grant also 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)

5) The grant also succeeded in exploring the ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery, which was published recently in eLife (Corey et al 2019)
Exploitation Route The details we reveal about the essential process of protein secretion and membrane protein insertion that in some cases are unique to bacteria may provide a route towards the development of novel antibiotics (see collaboration initiated with the Dundee drug discovery unit).

Our recent findings we anticipate will provide new information about fundamental aspects of biology.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.bristol.ac.uk/biochemistry/news/2016/bristol-and-leeds-collaboration-reveals-a-new-mechanism-for-protein-secretion.html
 
Description The work conducted in this grant allowed us to develop a robust assay for measuring ATP driven protein secretion through the bacterial Sec machinery - a potential target for the development of new antibiotics. We have indeed instigated a collaboration with the Dundee Drug discovery unit to do just that. The preliminary results are very promising and we are exploring a number of leads.
First Year Of Impact 2015
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Policy & public services

 
Description Wellcome Trust Senior Investigator Scheme
Amount £1,580,000 (GBP)
Funding ID 104632 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2014 
End 01/2020
 
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 the protein translocation machinery: The access to a neutron beam and the application of SANS will provide important information on the structure of the complex, and in particular on the nature of the associated lipids ( important for the func 
Organisation Institut Laue–Langevin
Country France 
Sector Academic/University 
PI Contribution Analysis of the Sec machinery using small angle neutron scattering (SANS): We are now employing SANS to help address the objectives of the proposal. SANS provides the means to analyse the structure of the machinery, allowing also the distinction between the lipids and protein components. We aim to use these methods to investigate how the structure of the lipid and protein components of the translocation channel complex are affected by the partner protein SecA. This information will help towards our understanding of the mechanism of protein secretion.
Collaborator Contribution Provision of neutron beam time and experimental support
Impact Botte, M., Zaccai, N. R., Nijeholt, J. L. À., Martin, R., Knoops, K., Papai, G., Zou, J., Deniaud, A., Karuppasamy, M., Jiang, Q., Roy, A. S., Schulten, K., Schultz, P., Rappsilber, J., Zaccai, G., Berger, I., Collinson, I., and Schaffitzel, C. (2016) A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion. Sci Rep. 6, 38399
Start Year 2011
 
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 SANS with Lise Arleth and Henrich Frielinghaus 
Organisation Julich Research Centre
Country Germany 
Sector Academic/University 
PI Contribution We provide of samples for analysis by SANS
Collaborator Contribution Analysis of our samples by SANS and data analysis
Impact Published a paper on the analysis of the structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon: see Martin, R., Larsen, A.H., Corey, R.A., Midtgaard, S.R., Frielinghaus, H., Schaffitzel, C., Arleth, A. and Collinson, I. (2019) Structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon: under revision Biophysical Journal - bioRxiv - https://doi.org/10.1101/490250
Start Year 2018
 
Description SANS with Lise Arleth and Henrich Frielinghaus 
Organisation University of Copenhagen
Department Niels Bohr Institute
Country Denmark 
Sector Academic/University 
PI Contribution We provide of samples for analysis by SANS
Collaborator Contribution Analysis of our samples by SANS and data analysis
Impact Published a paper on the analysis of the structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon: see Martin, R., Larsen, A.H., Corey, R.A., Midtgaard, S.R., Frielinghaus, H., Schaffitzel, C., Arleth, A. and Collinson, I. (2019) Structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon: under revision Biophysical Journal - bioRxiv - https://doi.org/10.1101/490250
Start Year 2018
 
Description Single molecule conductance measurements through the SecY complex 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Dr Ulrich Keysler, University of Cambridge Further single molecule analysis to study the protein translocation process
Collaborator Contribution The experiments failed
Impact None
Start Year 2013
 
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 School Week - Festival of Nature 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Inspirational contact with local school children

Appreciation and learning
Year(s) Of Engagement Activity 2014,2015
URL http://www.britishscienceassociation.org/events/general/bristol-festival-nature-2013