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“Fluctuograms” Reveal the Intermittent Intra-Protein Communication in Subtilisin Carlsberg and Correlate Mechanical Coupling with Co-Evolution

Figure 2

Mechanical coupling variation of subtilisin due to Ca²⁺ binding.

(a) Differences in the force constant of each residue between the Ca²⁺-bound and apo simulations of subtilisin as a function of time, (kcal/mol/Ų). Residues with large mechanical coupling variation are highlighted in the y-axis. See text for the definition of 's. (b) The location of the residues highlighted in (a) and (c). Residues specified by red fonts: residues have large mechanical coupling variation to Ca²⁺ binding, i.e. the average of 's is larger than 20 kcal/mol/Ų. Residues specified by red and boldfaced fonts: residues with large mechanical coupling variation and cover the mutation sites listed in (c) to within ±1. Residues specified by red and not boldfaced fonts: residues with large mechanical coupling variation but are not within ±1 of any of the mutation sites listed in (c). Residues specified by orange fonts: mutation sites listed in (c) with significant but not large mechanical coupling variation due to Ca²⁺ binding, i.e., the time average of 's is in between 10–20 kcal/mol/Ų. Residues specified by light blue fonts: mutation sites listed in (c) with medium or weak mechanical coupling variation, i.e., the time average of 's is less than 10 kcal/mol/Ų. (c) Mutation sites reported in protein engineering literature that can enhance the stability of subtilisin and the activity in a non-aqueous solvent. The residues are colored and boldfaced according to the criteria described in (b).

doi: https://doi.org/10.1371/journal.pcbi.1002023.g002