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. 2013 Jul 4;9(7):e1003129. doi: 10.1371/journal.pcbi.1003129

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© 2013 Song et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Briefly, FoldX followed by FEC (free energy calculation) are used to search for potential thermostable single mutants, from which a dozen are selected for experimental tests. The distribution of thermostable mutants is analyzed to identify the “hot spot”. Then more mutants in the “hot spot” are calculated by FEC and those predicted to be more stable are tested by experiments. Finally all stabilizing mutants are pooled together and multiple mutants are combined to further improve the protein's stability.