Hexaporins: the rational design of transmembrane channels

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.

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.

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.