Tertiary conformational changes of the Neurospora crassa plasma membrane H+-ATPase monitored by hydrogen/deuterium exchange kinetics: a Fourier transform infrared spectroscopy approach
1994
Goormaghtigh, E. | Vigneron, L. | Scarborough, G.A. | Ruysschaert, J.M.
Attenuated total reflection Fourier transform infrared spectroscopy of hydrated films of the Neurospora crassa plasma membrane H+-ATPase has been used to monitor the alpha-helix and beta-sheet contents and amide hydrogen exchange rates of the enzyme in the absence of ligands or locked in several stages of the enzyme catalytic cycle by MgADP, Mg-vanadate, and MgATP-vanadate. No difference larger than 2% was found in the alpha-helix or beta-sheet content of the H+-ATPase in different conformational states. However, when the rate of hydrogen/deuterium exchange monitored by the evolution of the area of amide II and amide II' is decomposed into three components, the number of amide proton characterized by a short exchange rate (1.1 min) fall from 38% of the protein amide protons (or 37% in the presence of Mg2+ alone) to 24-27% in the presence of Mg-vanadate and MgATP-vanadate and to 19% in the presence of MgADP. These results suggest that the conformational changes known to occur when the H+-ATPase interacts with the above ligands are predominantly tertiary structure changes.
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