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].

Protein melting: the Lindemann criterion applied to proteins

The full knowledge of the structural and dynamic properties of the native, transition, intermediate, and denatured states of proteins is a key goal of life and physical sciences. Indeed, this kind of information can lay the foundation for the deep understanding of folding and unfolding of proteins which, in turn, are crucial processes in the metabolism of cells, regulating biological activity and targeting proteins to different cellular locations. In our work (Katava et. al., PNAS (2017) here)we focus the attention on the sub-nanosecond dynamics of a model protein in correspondence of the melting transition, when it is embedded in three different solvents. We show that, although the different solvents modify the protein melting temperature, a common scaling toward a constant value for the local fluctuations is attained when approaching the unfolding temperature.
Quite remarkably, this result is reminiscent of the famous criterion for melting of solids proposed by F.A. Lindemann in 1910, which states that crystals liquefy when their atomic root mean square fluctuations exceed a certain threshold value. The common scaling we found for protein mean square displacements at melting not only sheds unique light on the relationship between protein flexibility and stability, but also opens the possibility to predict protein unfolding in special environments (e.g., the cell interior) by following thermal local fluctuations.
The striking analogy between the melting of inorganic crystals and native biomolecules suggests that these seemingly very different systems may share similar behavior in correspondence of phase transitions. On these grounds, we may speculate that simplified theories of solids can also be effectively applied to interpret the behavior of complex biological systems.

Strategies for the Design of Thermostable Enzymes in Targeted Mutagenesis

Flexible loops and mutations sites of E. Coli transketolase.
From the article in Scientific Reports 
(Scientific Reports 2017 7:41212 doi:10.1038/srep41212)

The relationship between protein flexibility and thermostability is still a foggy topic. However, if one assumes that increasing the rigidity improves enzymes thermostability, as described in many works, the rigidifying flexible sites (RFS) is one of the most straightforward strategies applicable in targeted mutagenesis.
Two possible cheap ways to identify residues which can be mutated to rigidify a protein loop are the “back to consensus” concept and the computational design based on ΔΔG calculations in Rosetta. This latter is powerful tool to predict stabilising variants, although it is not able to discriminate against mutations leading to activity loss. Conversely, the former, since it is based on the selection from functional mutations existing in natural variants of the enzyme, will more likely identify mutations preserving the catalytic function, but with more modest stability enhancements. For a practical application of these strategies and their comparison, see here.

Probing thermal activation of functional modes in proteins

Contribution of different modes to the overall protein internal motion is determined in the optimal working temperature regime of the protein.

The life of a protein is one of constant wiggling and reshuffling, i.e.  fluctuating and transitioning from one conformational substate to another. Untangling the correlated protein motions and targeting those that are functionally relevant represents a fundamental challenge.

We have recently successfully coupled Neutron Spin Echo spectroscopy to Molecular Dynamics simulations and theoretical modelling to individuate long-range functional modes. In the experiment, the protein is heated over a temperature range that includes the optimal working temperature, and the correlated protein motions in the nanometer and nanosecond regime are characterized in terms of their diffusion coefficients. Subsequently, molecular simulations and theoretical modelling are used to probe the contributions of different protein regions to the experimental signal. The functional modes are individuated as those activated in the optimal working temperature regime and being relevant in the functional context of the protein  (i.e. proximity to the active site, conservation...).

When the methodology was applied to a non-allosteric eukaryotic Lactate Dehydrogenase in its apo state, we discovered that the temperature increase results in a conformational change equivalent to that achieved as an effect of allosteric activators in allosteric bacterial LDHs, reflecting the richness of evolutionary pathways leading to the same functional configuration.

Enjoy our work 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.