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 .