Difference Between the E1 and E2 Conformations of Gastric H⁺/K⁺ -ATPase in a Multilamellar Lipid Film System: Characterization by Fluorescence and ATR-FTIR Spectroscopy Under a Continuous Buffer Flow

Author(s): Vander Stricht, Delphine, Vincent Raussens, Keith A. Oberg, Jean-Marie Ruysschaert, Erik Goormaghtigh

Journal: Eur. J. Biochem. (2001) 268: 2873-2880.

Abstract:
A liquid flow cell was used for an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) study of conformational changes taking place in the gastric H⁺/K⁺-ATPase. Shifting from E1 to E2 form is induced by replacing Na⁺ by K⁺ ions. Introducing ions through a flow passing over a protein multilayer film induced the conformational change without cell manipulations. Measurement sensitivity was thereby improved by about one order of magnitude. The detection threshold allowed the possibility to detect a change affecting five amino acids out of the 1324 that compose the H⁺/K⁺-ATPase molecule. It appeared that fewer than five amino-acid residues undergo a conformational change upon replacing Na⁺ by K⁺ ions in the medium. Evidence that conformational changes occur in an identical system was brought by monitoring the fluorescence of fluorescein isothiocyanate-labeled H⁺/K⁺-ATPase in similar conditions. Our data suggest that essentially the tertiary structure of the protein is modified.