Hexaporins: the rational design of transmembrane channels

Lead Research Organisation: University of Bristol
Department Name: Chemistry

Abstract

Our research is concerned with understanding how biology builds functional structures using molecular building blocks, such as nucleic acids (DNA and RNA) sugars, proteins and lipids. The latter two are the subjects of this grant proposal.

Protein molecules are polymers of amino acids that fold into defined three-dimensional (3D) functional structures. For example, collagen provides scaffolding in most of our tissues; haemoglobin transports oxygen from the lungs to active organs; and hexokinase breaks down glucose-containing foodstuffs to help provide energy in biology.

Many proteins fold and function in water. Essentially, there are two types of amino acid in proteins: hydrophobic ones, which are literally "water hating", and polar ones, which are soluble in water. A water-soluble protein with both polar and hydrophobic parts will fold to put most of its polar amino acids on its surface and in contact with water, and bury most of its hydrophobic amino acids.

However, much of biology goes on at the interfaces between, or within the membranes of cells, and these are not simple water-filled spaces, and a different set of proteins is needed.

Biological membranes surrounding cells are largely made up of lipid molecules. Lipids also have two distinct hydrophobic and polar regions. In membranes, many lipids aggregate together to form a bilayer, in which one leaf of lipids interacts with another burying the hydrophobic parts, leaving the polar parts exposed to water; much like in a sandwich with the bread (the polar parts in this analogy) on the outside, and the filling (the hydrophobic parts) in the middle. This organisation makes largely impermeable barriers, which presents a problem in biology, and other molecules, namely membrane-spanning proteins, are needed to facilitate transport and communication across the membrane. Nature uses these proteins to perform many functions, such as allowing nutrients into cells; excreting waste; exporting defence molecules; conveying signals across membranes; and even converting light into chemical energy.

Membrane-spanning proteins have a different overall chemistry to water-soluble proteins; they are hydrophobic on both the outside and the inside. This makes them more difficult to study, and harder to understand.

Recently, we discovered a new type of water-soluble protein structure, which we call CC-Hex. It has 6 protein chains, each of which folds up into a helix. These bundle to form a cylinder with a hole through it, a little like a stack of polo mints. This structure resembles membrane-spanning proteins called channels. Here, we propose to turn the water-soluble CC-Hex into a membrane-spanning protein by rational protein design. The key is that we understand both the chemistry and the structure of CC-Hex, which will guide our designs.

Why do this? The famous physicist Richard Feynman remarked that what he could not build, he did not understand. This is the principle that we have adopted: we will look at natural membrane-spanning proteins, learn from them, and then test our understanding by designing simplified membrane-spanning channels from CC-Hex. There is a risk that this might not work, but the potential rewards are high: we stand to learn how some of biology's components assemble at the very least; and possibly we could apply this understanding to create new proteins that might find applications in other areas of fundamental science and biotechnology.

For instance, a class of natural membrane-spanning channels known as the aquaporins transport and control the balance of water across cell membranes. As an example, in the kidneys aquaporins recover water from urine concentrating it to help avoid dehydration. Aquaporins are large complicated molecules. If we could capture their properties in a small protein like CC-Hex, we could possibly produce new molecules with potential application in water-purification and desalination devices.

Technical Summary

We aim to take a new and tractable peptide-design scaffold, CC-Hex, and engineer peptides and proteins that insert and assemble into membranes. These will then be tailored to make membrane-spanning channel proteins, "hexaporins", targeting water- and calcium-transport functions. This will require: rational peptide and protein design; new membrane-activity assays; combinatorial peptide synthesis; molecular biophysics; and structural biology.

Rational peptide design will involve mutating the central, outer faces of the CC-Hex helices to promote membrane insertion and assembly. This will be guided by bioinformatics and modelling, and tested via solid-phase peptide synthesis, followed by solution-phase biophysical characterisation and X-ray crystallography.

As with certain amphipathic helical peptides, e.g. antimicrobial peptides, there is the risk of general membrane disruption. To circumvent this potential problem, we will also design and produce single-chain, protein variants of CC-Hex and, subsequently, the hexaporins. This will build on our crystal structure of a Asp3His3-heterohexamer variant of CC-Hex. Proteins will be designed rationally, produced recombinantly and characterised by solution-phase biophysics and X-ray crystallography.

The peptides and proteins produced and the different activities being tested, require quick, simple and robust assays for both general and specific membrane activity. For these, we will build on the droplet-interface-bilayer (DIBs) methods developed by Bayley. In particular, we will introduce multiplexing to allow the high-throughput of peptides and tests; and colourimetric and other visual assays.

Finally, whilst rational and iterative designs should deliver membrane-inserting peptides and proteins, the design of functional channels is likely to require a combinatorial approach. For this, we will create focused libraries with different amino acids displayed within the lumens of the hexaporin channels.

Planned Impact

We will engage with audiences beyond our academic colleagues. We envisage two broad groups of beneficiaries, and propose to foster relationships as follows.

1. UK and international biotechnology industry
If we succeed in making hexaporins we envisage potential applications as components for biosensing, filtration and water-purification devices.

Regarding the provision of components for new water-purification systems, and in particular desalination, this would have a broad societal and economic impact. Energy efficient water purification is one of the grand challenges of this century. It has the potential to improve the lives of billions, particularly in the developing world and disaster-hit or drought-affected areas. However, there are significant materials and processing issues with current desalination systems that limit their efficiency. One aspect that our approach could contribute to this is at the molecular-design level of new water-selective filters. With an Australian collaborator, DNW noticed the analogy between the channel of our de novo protein structures, CC-Hex, and those of the aquaporins, some of which exclusively conduct water. However, the aquaporins are large, natural, multimeric membrane proteins, which makes them difficult to prepare, handle and engineer. Simplified, peptide analogues and peptidomimetics of the aquaporin channel would potentially ease production and engineering of filter components, and start to address problems associated with biodegradation and biofouling in desalination systems.

Though some way off realisation, initially we will explore possibilities for exploiting the hexaporins with our Australian collaborators, who include engineers, materials scientists, chemists and microbiologists, to determine if the hexaporins could provide or inspire new components for desalination systems. We anticipate that seeing any hexaporins through to such applications would take 10 - 25+ years.

2. UK public debate around Synthetic Biology
Synthetic Biology is an emerging area of research that combines engineering and biology. By applying engineering principles to biological systems, we may be able to re-design, or create from scratch, biological systems that perform new functions. This research potentially raises societal issues in terms of safety, security, regulation, ownership, and how to deliver maximum benefit of any emerging technologies. It is important that the public has a voice in the development of this exciting new field.

Both the Royal Academy of Engineering and the BBSRC have commissioned work that explores public attitudes to synthetic biology and its applications. Although public opinion is largely positive about the potential benefits of this work, it is imperative that researchers continue to talk about their research, its likely impacts and limitations, and to hear the concerns and interests of members of the public.

As outlined in our 'Pathways to Impact', we will continue to carry out public engagement activities to open up discussion about synthetic biology with a variety of audiences. Specifically, we will engage primary and secondary school pupils, teachers and adult members of the public in discussion and debate about this exciting area of research. We hope that through these interactions the public audience will be more positively disposed towards research in this area, and will have increased trust in the scientists that carry it out.

A second audience to be impacted through public engagement activities are the early career researchers in the Woolfson, Brady and Bayley labs in Bristol and Oxford. DNW and Gail Bartlett in particular will be involved in the planning and delivery of PE activities, receiving dedicated training from qualified professionals, as well as experiential training opportunities at the events themselves.

These aspects of our Impact plan are already in place, and we plan to continue PE activites for the next 5 years.

Publications

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Thomson AR (2014) Computational design of water-soluble a-helical barrels. in Science (New York, N.Y.)

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Woolfson DN (2015) De novo protein design: how do we expand into the universe of possible protein structures? in Current opinion in structural biology

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Niitsu A (2017) Membrane-spanning a-helical barrels as tractable protein-design targets. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Mahendran KR (2017) A monodisperse transmembrane a-helical peptide barrel. in Nature chemistry

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Heal JW (2018) Applying graph theory to protein structures: an Atlas of coiled coils. in Bioinformatics (Oxford, England)

 
Description We have discovered a small natural peptide that assemblies to form discrete, specific and stable channels in lipid bilayers (published in Nature Chem), and we have successfully designed other peptides completely from scratch that do similar things (in preparation for publication). These are being explored as components of novel biosensing systems.
Exploitation Route Yes. We have two PhD students and a post-doc actively working on and following up discoveries from these studies.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Title CCBuilder 
Description A computational method and GUI for design and engineering of coiled-coil assemblies of all oligomer states. 
Type Of Technology Software 
Year Produced 2014 
Open Source License? Yes  
Impact Being used and adopted by national and international research groups. 
URL http://coiledcoils.chm.bris.ac.uk/app/cc_builder/
 
Title ISAMBARD 
Description Builds parametric models of proteins for protein design. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact Becoming widely used by protein designers and engineers. 
URL http://woolfson-group.github.io/index.html
 
Description BBSRC PR: Designer barrel proteins 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Science publication, Computational design of water-soluble a-helical barrels [Science 24 October 2014: Vol. 346 no. 6208 pp. 485-488 DOI: 10.1126/science.1257452] was publicised on the BBSRC news page.

Unknown.
Year(s) Of Engagement Activity 2014
URL http://www.bbsrc.ac.uk/news/industrial-biotechnology/2014/141024-pr-bristol-team-creates-barrel-prot...
 
Description Parliamentary Science Committee presentation 2014 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Approximately 200 people attended the Parliamentary and Scientific Committee meeting on the 17th June 2014. The audience included Parliamentarians, members of scientific bodies, science-based industry and academics.
http://www.scienceinparliament.org.uk/sample-page/programme/
This meeting has subsequently been written up and included in the Autumn 2014 Science in Parliament (Vol 71 No 4: pgs 20 - 26) publication.

Unknown
Year(s) Of Engagement Activity 2014
URL http://www.scienceinparliament.org.uk/wp-content/uploads/2013/09/Autumn-Contents-page.pdf
 
Description Pint of Science Festival: Dark side of protein science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact As part of the Pint of Science Festival, researchers from BrisSynBio participated in the 'Dark Matters' event. The event was held in Friska café, Bristol, and involved scientific crafts and discussions between researchers and the public.
Director of BrisSynBio, Professor Dek Woolfson, along with Gail Bartlett, Jack Heal, Drew Thomson and Chris Wood organised the event 'Dark Matters'. Analogous to the idea of dark matter, protein science focuses on the protein structures that could theoretically exist but are not present in natural biology.
Year(s) Of Engagement Activity 2015
URL http://www.bristol.ac.uk/publicengagementstories/stories/2015/dark-side-protein-science.html
 
Description Pint of Science, Bristol, UK, May 2015, "From galaxies of stars to a new universe of proteins" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Part of the Pint of Science 2015 Programme in Bristol. About 60 people attended.
Year(s) Of Engagement Activity 2015
URL https://pintofscience.co.uk/event/dark-matters/
 
Description RSC: Synthetic Biology: The Free Edinburgh Festival Fringe Show (Heal) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Supported by an award from the Royal Society of Chemistry,the Edinburgh Fringe Festival hosted its first science stand-up on the subject of synthetic biology. Jack Heal's 'Do Scientists Dream of Synthetic Sheep?' show took a comedic approach to genome engineering, de-extinction and more - with the crowd helping to shape its direction with questions and discussion. The show considered questions from artificial life to Jurassic Park, and ran for 21 days.
Purpose: To interest the public in science.
Outcome: The comic felt freshly enthused about doing [synthetic biology] research.
Reflection: Free shows encourage people to take risks in their choices of which shows to see. This spirit is perfect for science outreach events which have to try hard to avoid becoming 'by scientists, for scientists'.

None yet.
Year(s) Of Engagement Activity 2014,2015
URL http://www.bristol.ac.uk/publicengagementstories/stories/2016/jack-heal.html?platform=hootsuite
 
Description Synthetic proteins for a synthetic biology: faster, fitter, stronger 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Better Humans Science Café, Bristol, UK, October 5 2016, "Synthetic proteins for a synthetic biology: faster, fitter, stronger"
Year(s) Of Engagement Activity 2016
 
Description The rise and rise of synthetic biology in the UK: science, policy and public perception 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Invited to Houses of Parliament, London, UK, June 2014, to speak to the Parliamentary and Scientific Committee.
Year(s) Of Engagement Activity 2014
URL http://www.scienceinparliament.org.uk/wp-content/uploads/2014/05/17-June-AGM-agenda.pdf
 
Description We the Curious, Bristol, UK, September 2019, Futures 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Woolfson gave an interactive talk on protein design and synthetic biology to a general as part of Bristol Futures 2019 at We the Curious, Bristol, UK, in September 2019.
Year(s) Of Engagement Activity 2019
URL https://www.futures2019.co.uk/events/we-the-curious/