Thermodynamic and structural effects of a single backbone hydrogen bond deletion in a metal-assembled helical bundle protein

Transition metal ion-assembled three-helix bundle proteins provide templates to investigate the thermodynamic and dynamic structural consequences of the deletion of a single backbone hydrogen bond from an alpha-helical architecture. This deletion does not perturb the steady-state secondary structure of the protein as measured by circular dichroism spectroscopy but does decrease the overall folding free energy by ca. 0.7 kcal/mol. We have used intraprotein electron transfer as a measure of the structure-sensitive dynamics of our system. The deletion of a single hydrogen bond in one of the helices of a three-helix bundle does not significantly change the measured electron-transfer rate. This is in agreement with "Greenpath" electron-transfer pathway calculations, which assume a constant and invariant structure for the architecture. Given the exponential dependence of electron-transfer rate on distance and that fluctuations in the intervening secondary structure will cause variations in electron donor-acceptor distances, the measured electron-transfer rate associated with hydrogen bond deletion allows us to calculate a differential dynamic structural fluctuation associated with hydrogen bond deletion of less than 0.6 Angstrom over the millisecond lime scale of the experiment.