Molecular dynamics simulations show α4-5 linker destabilization. (A) Snapshots of the α4-5 linker (blue) for WT and mutant at 37°C. The mutated site and Cys⁴⁷⁵ are represented in green and yellow, respectively. Ribbon representations show a disruption of the helix in the linker of the mutant, whereas the molecular surface representation shows an increase in solvent-exposed area of the Cys. (B) Quantitation of Cys⁴⁷⁵ exposure (▴) and linker helical content (▪) from molecular dynamics simulation. (inset) Normalized fraction of helical content along the backbones at 37°C. Note that compared with WT (blue line), the Q471P mutant (red line) shows considerable loss in helicity (approximately 40%) in the linker region. Arrows denote area of helix altered by proline mutation.