Tertiary conformational changes of the Neurospora crassa plasma membrane H(+)-ATPase monitored by hydrogen/deuterium exchange kinetics. A Fourier transformed infrared spectroscopy approach.
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 protons characterized by a short exchange rate (1.1 min) falls 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.
Laboratoire de Chimie Phsique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
E
E
Goormaghtigh
L
L
Vigneron
G A
GA
Scarborough
J M
JM
Ruysschaert
D000227:Adenine Nucleotides / Q000378:metabolism; D000577:Amides; D003903:Deuterium; D005583:Fourier Analysis; D007700:Kinetics; D008024:Ligands; D009492:Neurospora crassa / Q000201:enzymology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared
D000227:Adenine Nucleotides; D000577:Amides; D008024:Ligands; D003903:Deuterium; D006180:Proton-Translocating ATPases