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| 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.
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.
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