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 insideout. They were subjected to proteolysis by using Pronase, proteinase EL, trypsin, and carboxype...

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Bibliographic Details
Published in:The Journal of biological chemistry 1995-07, Vol.270 (30), p.17685-17696
Main Authors: Vigneron, L. (Universite Libre de Bruxelles, Brussels, Belgium.), Ruysschaert, J.M, Goormaghtigh, E
Format: Article
Language:English
Subjects:
ATPASA
ATPASE
Bacteriorhodopsins - metabolism
Cell Membrane - enzymology
ESTRUCTURA CELULAR
ESTRUCTURA QUIMICA
Fungal Proteins - chemistry
GLYCINE MAX
Hydrolysis
Membrane Proteins - chemistry
Membrane Proteins - metabolism
NEUROSPORA CRASSA
Neurospora crassa - enzymology
Peptides - chemistry
Peptides - metabolism
Protein Structure, Secondary
PROTEOLISIS
PROTEOLYSE
Proton-Translocating ATPases - chemistry
Proton-Translocating ATPases - metabolism
Spectroscopy, Fourier Transform Infrared
STRUCTURE CELLULAIRE
STRUCTURE CHIMIQUE
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Summary: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 insideout. They were subjected to proteolysis by using Pronase, proteinase EL, 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
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.270.30.17685