Graphical comparison from MD simulations of the C-lobe of apo-CaMs showing conformational changes in mutant CaMs with repacking of helices 2 and 3 and highly flexible Ca2+-binding site 4 in apo-CaMWT compared with the mutant CaMs, confirming the RMSF results. (A–C) Top clustered models of the C-lobe of apo-CaMF90L (red), apo-CaMF93L (yellow), and apo-CaMF142L (purple) are aligned with apo-CaMWT (green) in A, B, and C, respectively. Amino acid side chains of Phe90 (pink) and F90L (red) from apo-CaMWT and apo-CaMF90L (A), amino acid side chains of Phe93 (red) and F93L (yellow) from apo-CaMWT and apo-CaMF93L (B), and amino acid side chains of Phe142 (light blue) and F142L (light purple) from apo-CaMWT and apo-CaMF90L (C) are shown in the stick representation. Helix 3 from apo-CaMWT (magenta) resides at a larger angle in reference to the y axis compared with helices 3 from Phe → Leu mutant CaMs (dark blue) in A, B, and C, consistent with repacking of the structures. Quantification of the differences between apo-CaMWT and mutant CaMs shown in A, B, and C are summarized in Table S2. (D) Top 10 clustered models of apo-CaMWT aligned to represent an ensemble of possible states. Amino acid representation and color are conserved from A–C. Helix 3 is in purple. Helix 3 is mostly in a similar angle in reference to the y axis, with a much more extreme model present in the distribution. In addition, Ca2+-binding site 4 has a wide distribution of states, which does not align with those from the mutants (A–C). (E) Panel D rotated 180° to the right around the y axis.
