Fourier transform infrared spectroscopy study of the secondary structure of the reconstituted Neurospora crassa plasma membrane H(+)-ATPase and of its membrane-associated proteolytic peptides.
We reconstituted purified plasma membrane H(+)-ATPase from Neurospora crassa into soybean phospholipid vesicles (lipid/ATPase ratio of 5:1 w/w). The proteoliposomes contained an active ATPase, oriented inside-out. They were subjected to proteolysis by using Pronase, proteinase K, trypsin, and carboxypeptidase Y. Fourier transform infrared attenuated total reflection spectroscopy indicates that the amount of protein remaining after hydrolysis and elimination of the extramembrane domain of ATPase represents about 43% of the intact protein. The secondary structure of intact ATPase and of the membrane-associated domain of ATPase was determined by infrared spectroscopy. The membrane domain shows a typical alpha-helix and beta-sheet absorption. Polarized infrared spectroscopy reveals that the orientation of the helices is about perpendicular to the membrane. Amide hydrogen/deuterium exchange kinetics performed for the intact H(+)-ATPase and for the membrane-associated domain demonstrate that this part of ATPase shows less accessibility to the solvent than the entire protein but remains much more accessible to the solvent than bacteriorhodopsin membrane segments.
Vigneron
L
L
Laboratoire de Chimie Physique des Macromolecules aux Interfaces, Université Libre de Bruxelles, Belgium.
Ruysschaert
J M
JM
Goormaghtigh
E
E
D001436:Bacteriorhodopsins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D005656:Fungal Proteins; D008565:Membrane Proteins; D010455:Peptides; D001436:Bacteriorhodopsins; D006180:Proton-Translocating ATPases