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 template s to investigate the thermodynamic and dynamic structural consequences of t he deletion of a single backbone hydrogen bond from an alpha-helical archit ecture. This deletion does not perturb the steady-state secondary structure of the protein as measured by circular dichroism spectroscopy but does dec rease the overall folding free energy by ca. 0.7 kcal/mol. We have used int raprotein electron transfer as a measure of the structure-sensitive dynamic s of our system. The deletion of a single hydrogen bond in one of the helic es of a three-helix bundle does not significantly change the measured elect ron-transfer rate. This is in agreement with "Greenpath" electron-transfer pathway calculations, which assume a constant and invariant structure for t he architecture. Given the exponential dependence of electron-transfer rate on distance and that fluctuations in the intervening secondary structure w ill cause variations in electron donor-acceptor distances, the measured ele ctron-transfer rate associated with hydrogen bond deletion allows us to cal culate a differential dynamic structural fluctuation associated with hydrog en bond deletion of less than 0.6 Angstrom over the millisecond lime scale of the experiment.