The change in net fluctuations and correlations of the mutated residues for successive evolution of MR to GR proteins.

<p>(A) The change in net fluctuation between successive ancestral proteins, AncCR, AncGR1 and AncGR2 for mutated residues. Those residues identified as critical to alter-function are noted in red. The activation-function (AF) helix contains mutations 224 and 229. A cutoff (solid line) results in all critical mutations identified except for Y91C and L197M. Y27R is noted as critical to function but sites 65, 117, and 158 are false positives. (B) The cross correlation map with AncGR2 on the upper left and AncGR1 on the lower right. Circled in black are changes in the cross correlation associated with critical residues near the binding pocket. Squared in black are the changes in cross correlation due to critical mutation N26T forming a hydrogen bond with the AF-helix. Circles in white are additional changes in cross correlation not associated with critical mutations. (C) The cross correlations between the X and W mutations. The correlation between X and W mutations is higher for AncGR2, whereas AncGR1 X functional mutations are uncorrelated, increasing the flexibility in the binding pocket and allowing for promiscuous binding.