Dr Samuel Furse

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Sickle-cell anaemia and brittle bone disease are both caused by a single, small change in the structure of a protein. Cystic fibrosis is also caused by one of several ‘point’ mutations in the structure of just one protein. The question a scientist might ask is why this should occur—why should a small change in structure result in such a striking change in behaviour?

A practical difficulty in answering this question is how to test for the functional difference between normal and disease mutant forms of a protein. Cytosolic proteins can be characterised in aqueous systems that are effectively a model of the liquid part of cells. This means that the protein that is at fault in sickle cell anaemiacs can be characterised reasonably easily. The functional behaviour of the protein in cystic fibrosis is more difficult to characterise because it is a membrane protein rather than a cytosolic one.

Like lipids, membrane proteins have a part that likes water (hydrophillic) and one that does not (hydrophobic). This means that on contact with water without a membrane, they can re-fold, i.e. morph into a bit of a mess. The difficulty with a lot of membrane proteins—both disease and normal types—is that isolating them so that they can be characterised is an insurmountable obstacle.

The practical difficulty is therefore to isolate the protein in its native protein conformation. Detergents have been used for this purpose, and these do go some way to satisfying the amphiphilic character of membrane proteins. However, they can twist them in other conformers, and that can be just a difficult to understand, and just as useless in researching a disease. A better idea is surely to isolate the proteins with a patch of membrane that they like, such as the one they have naturally?

This is exactly what several groups of scientists have done [1-3]. The heart of this technique is to use a sort of molecular belt that acts like a polymerised lipid, with the greasy part of the lipid facing the fatty acid residues of the lipids and the hydrophilic part of the polymer facing the aqueous fraction. It turns out that this is thermodynamically stable and that sections of this polymerised lipid of the right length can behave like pastry cutters and chop out little discs of the membrane, with the all-important membrane protein nonchalantly sitting in the middle, like a prince in a sedan chair (Figure).

Figure. A section of the membrane is separated from the rest by the addition of co-polymer (yellow) to give a nanodisc (bottom).

Long et al. [1] showed that several mitochondrial proteins could be isolated this way. They established this by showing that the proteins isolated still had their enzyme activity after the isolation. This provided excellent data that functioning proteins resident in the membrane can be isolated for studies conducted in vitro.

Swainsbury et al. [2] tested different membrane protein isolation methods using a photoreaction centre from a purple bacterium called Rhodobacter sphaeroides. This protein complex is a useful tool for comparing methods because its behaviour with respect to light is dependent upon its conformation. In fact, both small and large differences in the conformation of the protein can be measured.

Doerr et al. [3] chose a different and more difficult target, an ion channel. Ion channels transport metal ions like sodium and potassium across membranes. The difficulty in isolating and characterising these is that unless they are in a membrane that divides two aqueous compartments, the changes in concentration of the ions cannot be measured. It is the molecular equivalent of letting someone in by opening the door of a room that has a wall missing. What this group therefore did was to isolate the ion channel protein complex (called KcsA) and reconstitute it in a membrane that did indeed divide two aqueous compartments. Electrophysiology techniques were then used to establish the activity of the KcsA they isolated. They found that indeed it was active after being both put into these little membrane patches (nanodiscs) and then reconstituted into another membrane.

What this research shows is that a variety of proteins, with different functions and from different species, can be isolated and characterised using this approach. The work is at an early stage, meaning that there is the unfortunate but predictable rush to secure the Intellectual Property rights. One form of this is a registered trade mark that has suffered from 90s-style marketing, having the name ‘lipodisqs^®’. This has also meant that the material for making nanodiscs has become commercially available. It is at a high price, the equivalent of £6m/Kg in the Netherlands.

However, as the facility of this technique becomes clear and more widely used, I predict that it will become easier to use and more finely tuned. We may yet see the characterisation of membrane proteins that exist in tiny quantities, such as those in individual neurone synapses. It may allow us to determine shifts in membrane protein concentration at different points in the cell division cycle. This technique may have scope to allow us to chart the shifts in molecular profile that represent the processes of a normal working body. However, its strongest application in the current zeitgeist may be to determine the functional difference between individual membrane proteins in diseased and healthy systems, enabling the characterisation of disease in a clearer and quicker way than ever before.

References

[1] A. R. Long, C. C. O’Brien, K. Malhotra, C. T. Schwall, A. D. Albert, A. Watts, N. N. Alder, BMC Biotechnology, 2013, 13, 41. DOI: 10.1186/1472-6750-13-41

[2] D. J. K. Swainsbury, S. Scheidelaar, R. van Grondelle, J. A. Killian, M. R. Jones, Angew. Chem. Int. Ed., 2014, 53, 11803. DOI: 10.1002/anie.201406412

[3] J. M. Doerr, M. C. Koorengevel, M. Schäfer, A. V. Prokofyev, S. Scheidelaar, E. A. W. van der Cruijsen, T. R. Dafforn, Marc Baldus, and J. A. Killian, Proc. Natl. Acad. Sci., 2014, DOI 10.1073/pnas.1416205112.

Conflict of Interest Statement: I share an office with the first author of [3]. The last author of the same paper is my boss, though I work on a separate project.

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A sturdy poetic metaphor for morning frost on grass is it being as dusted in sugar. This is a prescient analogy, given the data that is emerging about how organisms that survive the low temperatures, and desiccation.

How some living organisms can survive harsh cold or drought for extended periods, where others expire with only a modest drop in temperature or water availability, has been a puzzling question in biology for some time. There are several surprising examples–compare the story of some seeds that were found in the pages of a diary that had not been touched in the 17th century that were germinated and grown successfully at Kew Gardens, with a variety of fruiting plants that are susceptible to frost and need special attention when colder weather approaches. Summer fruit like plums are amongst the most susceptible.

There are also rather ordinary examples. A surprising number of the seeds with which we are familiar, are desiccated–sunflower, rape, mustard, hemp, sesame, poppy, hazelnut, walnut, acorn, and so on. The inescapable question is how they survive being put into what amounts to a static existence. Further questions come to mind almost immediately–how do cells in stasis know when to re-animate, how do they go about doing so once they have started? In order for this process to work, a number of individual cells must work in a co-ordinated way so that the organism can bounce back into active life in a controlled way.

The first question, of how being put into dry or frozen stasis without causing death, it is a good question from a molecular perspective as well as an ecological one. Lipids, and thus cell membrane studies, are central to this. We know from lipid physics studies that when the temperature falls below a certain point, the packing of the alkyl fraction of lipid bilayers changes in a way that heavily compromises its barrier properties. Significant drops in temperature can crystallise membranes.

A recent paper by Konov et al. [1] gives us some clues as to how scientists are beginning to understand resistance to these changes in lipid packing. In some recent work [1], they investigate the mediating effect of sugars (e.g. sucrose, trehalose) and sugar alcohols (e.g. sorbitol) on the behaviour of model lipid bilayers. They showed that these sorts of molecules stabilise the bilayers we see under ambient conditions, so that they remain in place at lower temperatures.

Figure. Trehalose (1, top left), surose (2, middle left) and sorbitol (3, bottom left), trehalose dibehenate (4, right).

What this group of authors conclude is that these sugars have the ability to replace the water molecules that interact with the bilayer under more ambient conditions. The molecular structure of the water and sugars are a clue to this (Figure). This means that the membrane behaves much more like it would do when water is present. This in turn has led to such molecules being called ‘cryoprotective’ reagents.

There are several biophysical techniques that could have be used to test the hypothesis that sugars are cryoprotective agents. In principle, any biophysical technique that can be used to indicate transitions between phases as a function of temperature could be suitable. A phase transition indicates a change in the way the lipids are assembled and thus moves away from the ‘normal’ membrane. In this work, they used a technique called Electron Spin Echo (ESE) Spectroscopy.

ESE is a relatively new technique. It is described as a pulsed version of Electron Paramagnetic Resonance, which means that it detects orientational vibrations (librations) on the nanosecond time scale [1,2]. It does this only in a particular type of molecular species, one with an unpaired electron, called a free radical. Specialised molecular probes, called nitroxide labelled stearic acid, are used in these systems at low concentrations. In this work [1], it has given clear evidence that certain molecules (sucrose, trehalose) are beer cryoprotective agents than others (sorbitol).

What is not yet clear is what happens on thawing, or in more complicated systems. In order to understand the full process of freezing, or desiccation, on an organism, what happens during re-animation is also required. We also have yet to know what happens to membrane systems that comprise membrane proteins, like ion channels or hormone receptors. The tools for research in the direction of controlled desiccation/freezing, and re-animation, may be closer at hand. A membrane-form of trehalose is commercially available, called trehalose dibehenate (Figure, R). The fatty acid residues on this sugar mean that it will be thermodynamically favourable for this species to be located in the membrane. This guarantees that a number of trehalose molecules will be at the surface of the membrane, irrespective of the prevailing conditions. The presence of such a species on the membrane may confer stability during desiccation and freezing.

The exciting thing about work in this field is that we know the process happens frequently in nature, and that understanding it is the first step to harnessing it, probably with incalculable results.

References

[1] K. B. Konov, N. P. Isaev, S. A. Dzuba, J. Phys. Chem. B, 2014, 118, 12478. DOI: 10.1021/jp508312n

[2] N. V. Ivanisenko, S. A. Dzuba, Appl. Magn. Reson., 2013, 44, 883−891. DOI 10.1007/s00723-012-0436-4.

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