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Tony Auffret's Blog.   

Can you build an inside-out protein?

A number of thoughts have been going around my head lately, all starting with a previous blog Most Proteins Don't Exist. The simple answer to “why most proteins don't exist” is that there is not enough matter in the known universe (read the blog if you don't believe me). This led me to wondering what is so special about the few proteins that do exist, and eventually to what might an extraterrestrial protein look like, if indeed such things exist.

Life on this planet arose because it is the only planet in our solar system in which water exists in all three phases of matter (see the Goldilocks Zone blog), which in a way argues that extra terrestrial proteins are unlikely; but it is still fun to speculate. If life arose because of water, then is it the properties of water which have selected the precious few proteins that exist? I think the answer to that has to be yes.

Nature has evolved with proteins which are active between two limits, the melting point of ice and the boiling point of water. Although life essentially does not exist outside these limits, it is hard to conceive that there would be any evolutionary disadvantage if a protein were capable of being active beyond these limits, but the truth is that, by and large, they are not.

Proteins are long linear molecules, but enzymes only exhibit useful biochemical properties when they are in a folded state. That folded state is not as stable as one might think and probably only exists because of the properties of liquid water. The entropic cost of ordering water molecules along a long linear chain must be quite high. Which makes one wonder “Do fibrous proteins coil together to reduce this entropic cost”? We are all familiar with the boiling of an egg, or in biochemical terms, if you heat a protein solution it unfolds and precipitates. What is not so familiar is that if you cool a protein solution sufficiently, it will unfold. The concept of unfrozen water below 0 °C may seem a little odd, but it is far from extraordinary. Small water droplets of water, clouds for example, can be cooled as low as – 40 °C without freezing and solutions can remain liquid below this temperature. The glass transition temperature (Tg') of a glycerol solution is about -100 °C, which means that there is liquid solution, albeit highly concentrated, at -80 °C, at -90 °C, in fact all all temperatures between -100 °C and the solution's boiling point.

The reason that low temperature protein unfolding is so little known is that, unlike high temperature unfolding, the protein does not aggregate. Nothing has changed about the protein, or indeed the unfolded state, but the water has changed. Cool water down from +20 °C to -20 °C, and it is 3 orders of magnitude less ionised. It is less polar and can solvate an unfolded protein!

It is this that leads me to ask the question “Can you build an inside-out protein”. If you can solvate the more hydrophobic amino acids on the outside of our hypothetical inside-out protein, what happens if you put the more polar residues inside? As most acid dissociations are exothermic, the van't Hoff equation tells us that acidic amino acid side chains will be less ionised at low temperature (the condition needed for solvating the outside). But does that mean basic amino acids would have a greater proton affinity? I am not sure, but of course such measurements are all made in aqueous solution, and we are looking at a solvent free environment inside our protein.

Gas phase (solvent free) measurements of proton affinity could be helpful, but as these are generally made in a mass spectrometer, so when considering “temperature” dependence we'd have to grapple with the concept of temperature in a vacuum. Perhaps crystal structure would be a more accessible guide. This is not the place to get into a consideration of the thermodynamics (temperature dependence) of crystal polymorphs (monotropic versus enantiomorphic relationships) but perhaps we can use the internal structure as an approximate guide to what is possible in a closely packed solvent free environment.

I haven't made an exhaustive search of amino acid crystal structures, access to the Cambridge Crystallographic Database, and published papers at €50 a copy are beyond the means of the “private” investigator. But aspartic acid, histidine and arginine crystals seem to exist in the zwitterionic form with uncharged side chains. The structure of an arginine crystal is particularity interesting as it suggests it is possible to have a network of hydrogen bonded amides not dissimilar to a β-pleated sheet (for a fee free preview see!divAbstract). One could also speculate that, in our inside-out protein, there is a reasonable likelihood of internal salt bridges, which by analogy to thermophile protein structures would suggest good thermal stability!

All of this presupposes that it is possible to cool water, suppress its ionisation, to such an extent that it will solvate hydrophobic amino acid side chains (the polypeptide backbone of course will still be polar) and there will be some thermodynamic driving force for the polar amino acids to be buried, presumably with an extensive hydrogen bonding network. Can this be done? I don't know. High pressure lowers the melting point of ice. At 200 MPa, (almost 2000 atmospheres) the melting point if ice is lowered to just below -20 °C and the homogenous nucleation limit (the temperature to which liquid water can, in theory, be cooled) to -90 °C. Could appropriate conditions exist in outer space? Probably not in something like an icy moon of Saturn; the subterranean ocean of Enceladus may be as hot as 93 °C, almost 300 °C warmer than the surface and much hotter than can be accounted for by tidal heating and radioactive decay. But elsewhere? A subsurface ocean in an exoplanet orbiting far from its own star?

Rather than speculate on the structure of extraterrestrial proteins, I would like to throw open the debate and issue a challenge for our readers to submit their views (on our LinkedIn page). The question is “can you build an inside-out protein”?

Why not let us know your thoughts on the subject?  You can join the discussion either on our Facebook page, in our LinkedIn group or via Twitter (see links iin the left hand panel above).

Missed our previous blog entries?  You can find them in our Blog Archive.

Tony Auffret also writes a regular blog article for taPrime Consulting.

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