Fourier transform infrared spectroscopy study of the secondary structure of the gastric H+,K+-ATPase and of its membrane-associated proteolytic peptides.
Membrane topology of the H+,K+-ATPase has been studied after proteolytic degradation of the protein by proteinase K. Proteinase K had access to either the cytoplasmic part of the protein or to both sides of the membrane. Fourier transform infrared attenuated total reflection spectroscopy indicated that membrane-associated domain of the protein represented about 55% of the native protein, meanwhile the cytoplasmic part represented only 27% of the protein. The secondary structure of the ATPase and of its membrane-associated domains was investigated by infrared spectroscopy. The secondary structure of the membrane-associated structures and of the entire protein was quite similar (alpha-helices, 35%; beta-sheets, 35%; turns, 20%; random, 15%). These data were in agreement with 10 alpha-helical transmembrane segments but suggested a participation of beta-sheet structures in the membrane-associated part of the protein. Polarized infrared spectroscopy indicated that the alpha-helices were oriented nearly perpendicular to the membrane plane. No preferential orientation could be attributed to the beta-sheets. Monitoring the amide hydrogen/deuterium exchange kinetics demonstrated that the membrane associated part of the ATPase molecule is characterized by a relatively high accessibility to the solvent, quite different from that observed for bacteriorhodopsin membrane segments.
Raussens
V
V
Laboratoire de Chimie-Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Brussels, Belgium.
Ruysschaert
J M
JM
Goormaghtigh
E
E
D000818:Animals; D019286:Endopeptidase K; D017506:H(+)-K(+)-Exchanging ATPase / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013270:Stomach / Q000201:enzymology*; D013552:Swine
D008565:Membrane Proteins; D010446:Peptide Fragments; D019286:Endopeptidase K; D017506:H(+)-K(+)-Exchanging ATPase