Understanding the Mechanism of Membrane Protein Insertion

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Bowen BJ (2020) Switching protein metalloporphyrin binding specificity by design from iron to fluorogenic zinc. in Chemical communications (Cambridge, England)

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Pope JR (2020) Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer. in Advanced science (Weinheim, Baden-Wurttemberg, Germany)

 
Description The grant is connected with grant BB/M003604/1 headed by Prof Ian Collinson. This grant had a postdoctoral researcher appointed for 18 months. The general secretory or 'Sec' machinery is responsible for protein translocation leading to secretion and membrane insertion. The Collinson Lab and others have advanced our understanding of the secretory process but membrane protein insertion through the Sec (or any other) machinery is very poorly understood. Therefore, the overarching aim is to understand the underlying molecular mechanism of protein insertion into membranes.

The availability of the bacterial holo-translocon (HTL) has been successfully used to reconstitute co-translational membrane protein insertion from purified components and for structure determination. To achieve the overarching aim, the project plans to exploit and extend these unique capabilities in a series of experiments exploring the structure and function of the membrane protein insertion apparatus.

The Jones aspect of the project was to incorporate non-natural amino acids (nnAA) into the components of the complex, notably the SecY component in preparation for functional analysis by the Collinson group. We focused on the incorporation of the photosensitive nnAA azido phenylalanine (azF). This aspect was achieved despite the researcher appointed to the project leaving early. Various different mutants were designed and generated followed by their recombinant production and purification followed by chemical modification with components such as fluorescence dyes. The Collinson group then used the mutant complexes for more detailed analysis. Initially we looked at the Sec YEG complex followed by the HTL. The later was to be performed in the Collinson lab.

By in large our aspect of the grant was delivered. The report for grant BB/M003604/1 needs to be referenced with regards to functional studies. The report of BB/M003604/1 will provide a more detailed outline of the new knowledge generation and new research questions opened up.

An additional benefit to come out of the grant is that we can now use nnAA to modify proteins for new applications and used, especially at the interface of biology with chemistry, nanoscience and materials science. As part of our engineering efforts we also generated a suite of additional proteins not related to Sec and HTL that contained nnAA such as azF and strained alkynes such as SCO with the aim of: (1) attaching proteins to nano materials and; (2) generating artificial protein oligomers through the use of Click Chemistry. Both these aspects were relatively successful resulting in several papers published or in preparation. We have used both Click chemistry and azF photochemistry to attach proteins to nano-carbon in designed many for optimal performance and communication. We have incorporated two nnAA that are bioorthogonally compatible (azF and SCO) into different monomers and linked them via Click chemistry. We have also used in silico design to help guide these approaches. This has in turn led to a number of new collaborations, notably with Nanoscientist, chemical biologists and endocrinologists. We believe that our new approaches developed will aid in the construction of novel biohybrid structures for use in nanoscience (including biosensing) and synthetic biology.
Exploitation Route From the perspective of the work carried out under the current grant, our findings will have the following impact:
1. Incorporation of new and useful chemistry into the functional centres of membrane proteins. This should aid our understanding of what are considered some of the hardest proteins to work with. The Collinson group has taken forward many of the Sec and HTL variants generated for more advanced studies.
2. Expansion of the use of genetically encoded nnAAs for uses outside of the normal biological context. We have developed approaches that will improve how we interface proteins with useful materials such as nano-carbon (CNTs and graphene) in defined and designed ways, overcoming one of the main challenges in the field.
3. Staff training. A researcher has been successfully trained on the processes by which to incorporate nnAAs into protein and to use the new chemistry inherent in the nnAA to probe protein function. This will be an important technical skills that will allow the next generation of scientists to use new approaches to engineer and probe biological system.
4. Collaboration. A new collaboration between Jones and Collinson has been formed.
Sectors Chemicals,Electronics,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Cardiff Synthetic Biology Initiative
Amount £45,908 (GBP)
Organisation SynbiCITE 
Sector Academic/University
Country United Kingdom
Start 05/2014 
End 09/2015
 
Description Dissecting the biomolecular basis of the action of Bcl3 interaction inhibitors.
Amount £44,955 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 02/2019
 
Description GW4 Accelerator award
Amount £71,189 (GBP)
Organisation GW4 
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 10/2016
 
Description Iraqi HCED
Amount £88,000 (GBP)
Organisation Iraqi Government 
Sector Public
Country Iraq
Start 10/2015 
End 10/2019
 
Title Control of protein activity through bioconjugation and light. 
Description The incorporation non-natural amino acids (nnAA) using a reprogrammed genetic code always new chemistry to be incorporated into proteins that can be used to modulate activity. We have used phenyl-azide chemistry as means to implement our strategy through the incorporation of the nnAA p-azido-phenylalanine. Two chemistries are available to control protein activity: (1) covalent bioconjugation with small non-biological chemical adducts; (2) irradiation to form a reactive nitrene radical that can follow different chemical routes. Both induce local conformational changes in the protein that can either up or down regulate protein activity. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact 1. We have now shared our plasmids with other groups around the world who wish to use our approach. 2. We now have several collaborations which include the exchange of researchers aimed primary at using the bioconjugation approach. 
 
Title Direct protein interfacing with carbon nano materials and DNA tiles. 
Description By incorporating non-natural amino acids into a protein at defined positions we can now precisely control assembly of hybrid protein materials. We have so far demonstrated the approach with two systems: (1) site-specific attachment of ssDNA to provide addressable assembly on DNA origami tiles. Using a non-biological reaction handle incorporated using a reprogrammed genetic code, ssDNA can be site-specifically attached to the protein of interest (POI) at an optimal position. The hybrid ssDNA-protein system can then be assembled on base DNA tiles with the ssDNA acting as the addressable element and the protein the active component. We have used this system to assemble multiple proteins on a single tile. When assembled, the proteins can show enhanced activity. (2) site specific attachment of proteins to carbon nano materials such as single walled carbon nanotubes (swCNTs) and graphene. Using a non-biological reaction handle incorporated using a reprogrammed genetic code, we have successfully attached proteins to these carbon nanomaterials to get functional communication between proteins. We have achieved this is two ways. (i) attachment of conjugates that allow non-covalent interfacing with the pi electron system of the carbon nanomaterials. (ii) direct covalent attachment using light activated chemical groups incorporated into the protein. We assembled, we have seen communication between the protein and carbon nano materials. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact 1. New ways of working. 2. Collaborations with other institutions including University of Southampton, Manchester and Queen Mary. We are jointly exploring how to exploit the new approach to nanoassembly. 
 
Title New approaches to designed protein oligomerisation. 
Description One of the most important events a protein will undergo is associating with itself or other proteins to from a functional complex; this is known as oligomerisation. Protein oligomerisation is common place in nature, with the majority of cellular proteins existing either permanently or transiently as oligomers. Oligomerisation is normally cooperative and synergistic in that properties such as function and stability are greatly enhanced compared to the monomeric form, and new properties can emerge (e.g. functional enhancement, switching); there is normally communication between individual monomer units that leads to these new or enhanced properties. The method involves taking normally monomeric proteins and then designing, building and testing new oligomeric protein species. While oligomerisation could be considered desirable, it is difficult to engineer into functional monomeric proteins due to the complexity of natural protein-protein interfaces. To meet this challenge, we used a new synthetic biology approach for generating protein oligomers: biorthongonal Click crosslinking using a reprogrammed genetic code to incorporate non-natural amino acids (nnAA) at designated residues in a target protein. At least two different types of nnAA were used that are not found in nature but can react on a 1 to 1 basis to form a defined crosslink. Using this system, we can explore the construction of dimer, trimers and beyond composed of identical and mixed protein units to generate a myriad of new structures of potential fundamental and technological use. To date the research has focus on autofluorescent proteins (e.g. GFP) but will quickly move on to constructing multi-enzyme complexes. This interdisciplinary project has additional focus on in silico protein design and engineering (e.g. protein docking simulations) and combines elements chemistry and biophysics. Techniques that embrace the overall concept. Primary: computational analysis, synthetic biology (reprogrammed genetic code systems). Secondary associated techniques: molecular biology (cloning and mutagenesis), protein chemistry (purification and analysis), protein 3D structure determination, biophysical analysis (various spectroscopy methods, including single molecule analysis). 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact This is an nascent project with the aim of generating communicating protein complexes akin to those found in nature. Our early designs and constructed systems show great promise including new protein structural scaffolds, functional synergy and new emergent functions. 
 
Title Site-specific protein modification through the use of non-natural amino acids. 
Description We have used expanded genetic code systems to incorporate new chemistry not present in nature with the aim to link protein to non-biological components ranging from small molecules to materials to other biomolecules. We have focused primarily on the use of phenyl azide chemistry to: (1) photo control protein activity; (2) link proteins to oligonucleotides for bottom-up assembly; (3) add small molecule adducts that regulate protein activity; (4) link proteins to materials such as graphene and carbon nanotubes. 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact We have used the approach to demonstrate the core aspects above namely (1) photo control protein activity; (2) link proteins to oligonucleotides for bottom-up assembly; (3) add small molecule adducts that regulate protein activity; (4) link proteins to materials such as graphene and carbon nanotubes. As stated above. 
 
Description Designed protein-carbon nanotube interfacing. 
Organisation Queen Mary University of London
Department School of Biological and Chemical Science QMUL
Country United Kingdom 
Sector Academic/University 
PI Contribution We have engineered a series of proteins ranging from autofluorescent proteins to antibiotic inhibiting proteins to contain a new reaction handle at specific sites. This new reaction handle, azidophenylalanine (azF) can used to interface proteins with CNTs using Click Chemistry.
Collaborator Contribution The collaborator, Dr Matteo Palma, is an expert in CNTs and their use including in terms of their application to sensing. They provide the CNT materials and analysis approaches to the project.
Impact 1 paper: see Freeley, Worthy et al in the main list. 1 grant in preparation: we plan on submitting a grant concerning the construction of novel biosensors. Multidisciplinary: the discipline involved include synthetic biology, protein engineering and design, biochemical/biophysical analysis, nanoscience, materials science, chemistry and physics.
Start Year 2016
 
Description Designed protein-nano carbon and protein-protein interfacing. 
Organisation Cardiff University
Department School of Pharmacy and Pharmaceutical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated engineered variants of fluorescent proteins that can be photochemically linked to nano-carbon or linked together to generate novel communicating oligomers.
Collaborator Contribution The partners, namely Dr Oliver Castell, is an expert in single molecule imaging using total internal resonance fluorescence microcopy (TIRFM). Through our collaboration, Dr Castell has undertaken extensive experimental measurement and analysis of the data as a contribution to the project.
Impact Outputs: 1 paper submitted and 1 paper manuscript in preparation. This is multidisciplinary project involving protein engineering and synthetic biology together with physics (single molecule measurements and analysis)
Start Year 2017
 
Description Developing a novel biosensor technology for biomedical applications 
Organisation University of Bath
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in fluorescent protein engineering. Clones and reagents for use in project.
Collaborator Contribution This was a four person collaboration at 4 different institutions. Each member bought their own expertise related to biomolecule sensing and imaging.
Impact The collaboration has just finished and we are awaiting finalisation of output. Briefly: So these data have been a long time coming. The project was successful in demonstrating of the edge shift effect on whole cells by microscopy, including in mouse cortical neuron treated with fluorescein labelled Amyloid Beta. The cells were imaged at a variety of different excitation wavelengths and extracted the full emission spectra. From here we can plot the centre of spectral mass (CSM; middle panel). We very see an edge shift effect associated with the treated cells. For this system, the observation of a significant edge shift seems to reflect A-Beta aggregation (confirmed by a raft of in vitro experiments). We find no major aggregation on cell bodies (dark blue), but much more significant on axons (cyan). Looking at the top right image, it actually looks like we might have the most aggregation at the synapses (red). We are still working on the data processing [Bath]. We are looking to have an MS together towards the end of the month and I hope you are all happy to have authorship. I would like to write something quite short, but I would be curious as to where people think is best for this story.
Start Year 2015
 
Description Precision attachment of proteins in DNA origami tiles through Click chemistry 
Organisation University of Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution a synthetic biology approach to modify (conjugate) a target protein in a pre-defined, truly orthogonal, biocompatible manner. Used to attached ssDNA to various proteins of interest.
Collaborator Contribution assembly based on DNA origami approaches including synthetic modification of DNA
Impact This is a multidisciplinary collaboration. We have currently submitted a paper and are in the process of writing grants. One has been submitted on protein-based assembly of enzyme systems but was rejected.
Start Year 2014
 
Description Protein engineering for new biosensor implementation. Director of Synthetic Biology. 
Organisation Molecular Warehouse Ltd
Country United Kingdom 
Sector Private 
PI Contribution The research background of myself (Dr Dafydd Jones) has mean that a small start-up company, Molecular Warehouse, are invited me to be their Director of Synthetic Biology.
Collaborator Contribution Due to the commercially sensitive nature of this work, I will not state any details. The partners, Molecular Warehouse, wish me to join their company on a part-time basis initially as their director of synthetic biology. This is primarily to lead their protein engineering and production efforts.
Impact There are no outputs as of yet
Start Year 2017
 
Description Structure and drug-based design of bcl3 and its complexes. 
Organisation Tenovus Cancer Care
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution My team generate recombinant protein and analyse bcl3 and its complexes. This includes structural analysis and the effect of drugs on complex stability. The collaboration involves members of Cardiff's School of Chemistry (Burma), School of Pharmacy (Westwall and Brancale) and Medicine (Rizzkallah). My group has gained funding from Tenovus Cancer research, KESS2 and Wellcome.
Collaborator Contribution Our partners supply drug compounds and in vivo test data.
Impact Funded Wellcome ISSF grant for 1 postdoc for 1 year. Funded KESS2 PhD studentship for 36 months in collaboration with Tenovus.
Start Year 2017
 
Description Structure and drug-based design of bcl3 and its complexes. 
Organisation Wellcome Trust
Department Wellcome Trust Institutional Strategic Support Fund
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution My team generate recombinant protein and analyse bcl3 and its complexes. This includes structural analysis and the effect of drugs on complex stability. The collaboration involves members of Cardiff's School of Chemistry (Burma), School of Pharmacy (Westwall and Brancale) and Medicine (Rizzkallah). My group has gained funding from Tenovus Cancer research, KESS2 and Wellcome.
Collaborator Contribution Our partners supply drug compounds and in vivo test data.
Impact Funded Wellcome ISSF grant for 1 postdoc for 1 year. Funded KESS2 PhD studentship for 36 months in collaboration with Tenovus.
Start Year 2017
 
Description Invited speaker at conferences. These include RSC Chemistry in the New World of Bioengineering and Synthetic Biology; RSC Chemistry and Biology Interface; RSC Bioorganic Firbush Meeting: Protein engineering and directed evolution; Synthetic biology in Pharma; European Society of Bio-organic Chemistry meeting; SWSB Structural Biology meeting; SCN Synthetic Biology symposium; Zing International Structure Biology and Drug discovery conference; Synthetic Biology Symposium in Glasgow; Harden Confer 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I have talked at many different conferences presenting work related to the funded work.

Making contacts and advertising the work supported by the grants.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013
 
Description School Visit (Wales). These include (i) Wales Gene Park sixth form conference (Nov 2012) to ~1600 A- level students at St David's Hall, Cardiff; (ii) visit to local sixth form to discuss genetic modification; (iii) visit to local primary school to talk and demonstrate why proteins are useful (e.g. washing powders and cheese making). (iv) talk at Coleg Gwent to A-level students concerning synthetic biology. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact talk and discussion with students

Informed students on current science
Year(s) Of Engagement Activity 2009,2015