Hexaporins: the rational design of transmembrane channels

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry


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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.


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

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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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Howorka S (2016) NANOTECHNOLOGY. Changing of the guard. in Science (New York, N.Y.)

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Pliotas C (2015) The role of lipids in mechanosensation. in Nature structural & molecular biology

Description Autonomous monodisperse transmembrane a-helix barrels have not been characterized previously. We have formed a functional transmembrane pore from 35-amino-acid a-helical peptides based on the C-terminal D4 domain of the E. coli polysaccharide transporter Wza. By single-channel current recording, we define discrete assembly intermediates and show that the pore is a barrel containing eight D4 peptides arranged in parallel. We suggest that an understanding of a-helix barrels illuminates the action of certain antimicrobial peptides and that engineered barrels will find applications in nanopore technology.
Exploitation Route We suggest that an understanding of a-helix barrels illuminates the action of certain antimicrobial peptides and that engineered barrels will find applications in nanopore technology.
Sectors Healthcare

Pharmaceuticals and Medical Biotechnology

URL http://bayley.chem.ox.ac.uk/