Mechanics of thermal adaptation

Somero’s corresponding state principle relates protein enhanced thermal stability with mechanical rigidity. A natural way to test the mechanical stiffness of a protein is to apply a directional force as in single molecule AFM experiments. Recent experiments have been therefore inspired by the possible correlation among the mechanical and thermal stabilities. Unfortunately single molecule experiments lack molecular resolution, and in silico realisation of thermal and mechanical unfolding can provide very useful insights. This is exactly what we have done in a recent work focused on two homologues belonging to the Cold Shock Protein family. Our results show that for these species there is not a correlation among the thermal resistance of the thermophilic Csp and its mechanical stability. The paper is out in JPC Letter [here].

Evolution and Thermoadaptation in Enzymes

How have enzymes evolved since life appeared on Earth? What has driven the adaptation of enzyme catalysis to different temperatures? Although massive work has been done, in 2017 these are still “hot” questions searching for answers. An interesting paper by Nguyen et al., just appeared on Science, tackled this unsolved issue by investigating the molecular mechanisms underlying thermoadaptation of enzyme catalysis through ancestral sequence reconstruction spanning 3 billion years of evolution, and using as a study-case the adenylate kinase (Adk). The authors assumed as true the well-supported hot-start hypothesis, which implies that life adapted to cooler temperatures because of the Earth’s cooling. According to this, a thermophilic enzyme had to adapt to maintain a high catalytic activity even at lower temperatures, while accommodating relaxed selection on thermostability. It has been hypothesized that enzymes overcame this thermal kinetic hurdle by reducing the enthalpic activation barrier. However, Nguyen et al., by reconstructing eight nodes of the Adk lineage and expressing them together with four modern Adk enzymes, found out something different. Indeed, from the analysis of the Eyring plots, they showed that the oldest ancestors had a strongly negative change in heat capacity of activation, which can explain their extreme slow catalysis at low temperatures. Conversely, along the thermoadaptation process toward cooler temperatures, this kinetic obstacle has been progressively removed, bringing the heat capacity of activation to zero. This close to zero heat capacity of activation was also observed for thermophilic enzymes evolved from mesophilic ancestors, but not for modern hyperthermophiles that remained thermophilic throughout their evolutionary pathway. This represents also a prove of the "evolutionary memory" of enzymes. To find out more about this new scenario, see here for the full manuscript. 

Interface matters: The stiffness route to stability of a thermophilic tetrameric malate dehydrogenase

Understanding the relation between protein flexibility, stability and function remains one of the most challenging, open questions in biophysical chemistry. For example, proteins need to be flexible to facilitate substrate binding but locally rigid to sustain substrate specificity. Enzymes from microorganisms that thrive at elevated temperatures, also referred to as thermophiles, are a natural study-case to dig into the issue. These proteins are stable and functional at a high temperature regime but generally lack activity at ambient conditions. Therefore, their thermal stability has been correlated to enhanced mechanical rigidity through the so-called corresponding states paradigm introduced years ago by Somero. The generality of this view, however, has been questioned by a number of experimental and computational studies. Computer simulations based on the molecular dynamics technique offer a unique opportunity to explore the correlation among mechanical rigidity and thermophilicity. In our recently published article in PLoS One we consider the specific case of two tetrameric orthologous malate dehydrogenase proteins from two bacteria that grow optimally at different temperatures. For these orthologues, as for other oligomeric proteins, the role of interfacial interactions becomes critical, adding up to the other cohesive forces acting on monomeric proteins. How the protein rigidity/flexibility patterns influence the stability and function of the two molecules is discussed in detail in the paper.

Dehydrogenases are proteins that require a cofactor in order to catalyze a reaction.
The picture above depicts one monomer of a lactate dehydrogenase in a
cofactor-bound (holoprotein) and a cofactor-unbound (apoprotein) form.