Darwin meets Gibbs: making a temperature-resistant protein

In order to perform its function, a protein should be properly folded. Therefore stability of this protein's native state is crucial for its function. Denatured protein can be toxic for the cell and requires specialised machinery to degrade it, thus compromising the cell's fitness. Having a denatured protein is not equal to just not having a functional one, it is equal to not having a functional one and having some costly junk.

Since stability is so crucial for protein function, it must leave its trace in the patterns of amino acid conservation. Bioinformatic studies show that there is a strong correlation between the Surface Accessible Area (SAA) of the residue and its conservation, or, simply speaking, conserved residues are mostly buried inside the protein. That sounds logical – the core should be more important for protein stability than its outer shell. The outer residues, on the other hand, can be rearranged in order to change the protein's binding selectivity and evolve new function. But lets get back to the core.

Basic thermodynamics relationships link protein stability to parameters like Gibbs free energy (ΔG), enthalpy (ΔH), entropy (ΔS), heat capacity (C, or, to be specific heat capacity at constant pressure, C_p) and absolute temperature (T). And adaptation to extreme temperatures gives us a striking example of thermodynamics shaping protein evolution. But first let us start with some basic theory - I know that sounds painful, but please stay with me for a moment!

Gibbs free energy is divided into enthaplic and entropic components (ΔG = ΔH - TΔS). By the definition of Gibbs energy, in order for the protein to be stable, ΔG of folding should be negative, and when it is positive, the protein unfolds.

Both of the components of ΔG change with temperature. Enthalpy changes linearly, with the proportionality coefficient being heat capacity (ΔC_p, ΔH(T) = ΔH(T₀) + ΔC_p(T-T₀)). Heat capacity is the amount of heat needed to change the temperature of protein by one degree.

Entropy also changes with temperature, though in a bit more complex way (ΔS = ΔS(T₀) + ΔC_pln(T/T₀)). When we combine the two components of ΔG, we get this:

ΔG(T) = ΔG(T₀) + ΔC_p(T-T₀) - ΔC_pTln(T/T₀)

This is a very interesting relationship. It gives ΔG(T) its characteristic shape with a maximum corresponding to the T of maximums stability, and two denaturation temperatures (on the graph below I plot –ΔG, rather than ΔG just so that the plot looks nicer).

We are all familiar with protein denaturation at high temperatures (we all boiled eggs!), but at lower temperatures? Well, this one happens as well, but very, very slowly, as all the reactions tend to at low temperatures, so we do not notice it that much. However, indeed, some proteins are better off when stored at -20C^o, than at -80C^o.

Heat capacity is intimately linked to the above-mentioned solvent accessible area (SAA). The reason for that is that it is the water surrounding the protein that gives it its heat capacity. Water molecules next to the protein are restricted in their freedom; they are essentially frozen, and ice, as we know, has tremendous heat capacity. When the protein denatures, its SAA increases, and so does the heat capacity. Heat capacity change upon denaturation in turn is determining the shape of folding ΔG dependence on temperature (see equation above).

And now we are primed to discuss how the extrermophylic proteins cope with high temperatures. One can imagine two obvious solutions. First, they could increase their stability (ΔG) (curve A). However, this would result in a bit too stable proteins that will be very hard to degrade, and this is not good for metabolism. Also, they will be too rigid, and flexibility is necessary for protein function. Second, they could move their temperature of maximum stability (curve B).

In reality they do something completely different! They decrease the ΔC_p instead, flattening the ΔG curve.

So how do they decrease the ΔC_p? Well this is all about the nature of the denatured state. ΔC_p is proportional to ΔSAA of protein unfolding, but proportionality is different for hydrophobic residues (these freeze water well, thus proportionality coefficient is high) and hydrophilic ones (these are similar to water in their nature, and thus do not restrict its movement too much, and the proportionality coefficient is lower).

Thermophilic proteins enrich the normally hydrophobic protein core with polar residues, forming salt bridges and dipole-dipole pairs. This results in a more rigid structure, thus you still pay in flexibility somewhat, therefore if there is no need for extreme temperatures, hydrophobic core is better.

Modifying the protein core is not an easy task since you need to compensate for one substitution with another (say, you have a positively charged residue, and now in order to compensate it you need a negatively charged one). Moving in the other direction (thermophilic to mesophilic) would be equally tricky. Therefore keeping your temperature stable – just like we do! – allows avoiding all these complicated thermodynamic matters altogether.

References:

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Drummond DA, & Wilke CO (2008). Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell, 134 (2), 341-52 PMID: 18662548

Geiler-Samerotte KA, Dion MF, Budnik BA, Wang SM, Hartl DL, & Drummond DA (2011). Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast. Proceedings of the National Academy of Sciences of the United States of America, 108 (2), 680-5 PMID: 21187411

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