Over 100°C

What factors contribute to protein stability at very extreme temperatures? What gain comes from entropy and enthalpy? And how to account for the delicate effect of temperature on molecular interactions like the hydrophobic and ionic ones? All this is tackled in a very intriguing work by Y. Matsura et al. "Thermodynamics of protein denaturation at temperature over 100°C: CutA1 mutant proteins substituted with hydrophobic and charged residues" recently published in Scientific Reports [see here]. By designing sequential mutations the authors were able to construct hyper-stable versions of the CutA1 protein and to extract the main thermodynamic parameters characterising their thermal stability. It is a very important work challenging both technical biochemical problems, like the aggregation of proteins generally occurring above 80°C, and the basic thermodynamics controlling protein stability over 100°C where for instance hydrophobic interactions cease to be entropically driven and ion-pairing can benefit from water dielectric constant decrease.  

There cannot be only one

Is it hydrophobics or electrostatics? Is it in structure or in dynamics? Is it an enhanced rigidity or an increased flexibility of the protein matrix? Maybe the answer is in water?

Relevant scientists might not agree on what factor plays the most important role in increasing thermal stability of (hyper)thermophilic proteins, but they all agree that not only one is overall responsible. The enhanced thermal stability of a thermophilic protein is usually a result of a well-orchestrated symphony of more than one structural and/or dynamical factors. 

Even so, several experiments have demonstrated that, in some cases, single point mutations are capable of increasing the thermal stability of an enzyme. Whenever that is possible, it does come in handy, since a thermophilic enzyme with the desired properties doesn’t always exist or even if it exists it is not trivial to obtain. So we go back to studying how thermophilic proteins are mastering it. After all, it gets down to identifying trends that are immediately applicable for a rational design. 

Such a useful trend was recently presented by H. Gohlke and coworkers advocating for the importance of “qualitative” hydrophobic contacts on protein stability. By qualitative contacts the authors mean - and effectively demonstrate - that it is not the size of clusters of hydrophobic residues that distinguishes (hyper)thermophilic proteins from their mesophilic homologues. It is rather the fact that thermophilic, and even more hyperthermophilic proteins, are enriched in those hydrophobic contacts that have a low (favorable) energy. With this, they achieve in distinguishing thermophilic over mesophilic proteins with a discrimination accuracy of 80%, something that is not achieved as well when they use other energy components such as hydrogen bond energy for example. 

Finally and most importantly, the authors successfully locate weak spots on three different proteins where mutations will lead to an increased thermal stability, as well as non-weak spots that should not be mutated as they already stabilize the protein. Moreover, the computational efficiency with which this can be done makes the method a potentially very useful tool for protein design.

A droplet of water forms a spherical shape,
minimizing contact with the hydrophobic leaf.
Photo taken by tanakawho