Structural difference in the H<sup>+</sup>,K<sup>+</sup>‐ATPase between the E1 and E2 conformations

Raussens, Vincent; Pézolet, Michel; Ruysschaert, Jean Marie; Goormaghtigh, Erik

doi:10.1046/j.1432-1327.1999.00365.x

Cited by 8 publications

(12 citation statements)

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“…7). In studying the substitution of cations in ATPase, Raussens et al (52) noticed that structural changes in the protein secondary structure result in a small band shift. In addition and as shown in the deconvoluted spectrum of the protein-birnessite complex, three new bands appeared at 1641, 1619, and 1548 cm −1 (Fig.…”

Section: Ftir Analysismentioning

confidence: 99%

Formation of Protein–Birnessite Complex: XRD, FTIR, and AFM Analysis

Naidja

Liu

Huang

2002

Journal of Colloid and Interface Science

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Section: Ftir Analysismentioning

confidence: 99%

Formation of Protein–Birnessite Complex: XRD, FTIR, and AFM Analysis

Naidja

Liu

Huang

2002

Journal of Colloid and Interface Science

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“…Because the hog gastric H + /K + ‐ATPase contains 1324 amino‐acid residues, a conformational change affecting 40–65 amino‐acid residues could go unnoticed with the sensitivity obtained in previous works [9]. Here, we report an ATR‐FTIR study of the gastric H + /K + ‐ATPase conformational change with a sensitivity improved by about one order of magnitude.…”

Section: Discussionmentioning

confidence: 74%

“…Many groups have attempted to determine the nature of the E1‐E2 conformational change in the P‐ATPases. Several techniques, such as CD, Raman and IR spectroscopy have been used [3,9,14–16]. While most of these studies concluded that the conformational change is small [3,9,14,16], with the notable exception of Gresalfi and Wallace [15], their sensitivity was limited to a few percent of the total signal intensity (typically 5% [9]) because of inherent limitations of the method used.…”

Section: Discussionmentioning

confidence: 99%

“…Raussens et al . [9] studied conformational changes induced by K + in the gastric H + /K + ‐ATPase by UV‐CD, Raman and ATR‐FTIR techniques. The difference between E1 and E2 spectra did not reveal any significant feature within the reproducibility limits of these types of experiment, indicating that structural differences between the E1 and E2 conformation are too small to be detected.…”

mentioning

confidence: 99%

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Difference between the E1 and E2 conformations of gastric H⁺/K⁺‐ATPase in a multilamellar lipid film system

Stricht

Raussens

Oberg

et al. 2001

European Journal of Biochemistry

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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.

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“…In contrast, Saccomani and others have suggested an increase in the a helical content upon addition of ATP, Mg 21 and K 1 [10]. Recently, Raussens and coworkers [11] were unable to observe structural changes by FTIR spectroscopy; they assumed that any changes were below the detection threshold (estimated to be 66 amino-acid residues) of the method.…”

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confidence: 97%

Monitoring of secondary and tertiary structure changes in the gastric H⁺/K⁺‐ATPase by infrared spectroscopy

Scheirlinckx

Buchet

Ruysschaert

et al. 2001

European Journal of Biochemistry

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Conformational changes occurring in the catalytic cycle of the H 1 /K 1 -ATPase were monitored by Fourier transform infrared spectroscopy (FTIR). Caged compounds were used to release ATP, in the presence of Ca 21 , to induce the transition between the E1 and E1-P conformation of the H 1 /K 1 -ATPase. In addition to bands associated with the photolysis of the caged compounds, some peaks of the difference infrared spectra were associated with changes in secondary structure and modifications of the ionization in the side chains of amino-acid residues (Glu or Asp). These changes were specific to the reaction between the ligand and the enzyme. We estimated that 39 amino acids changed their secondary structure during the reaction and four amino-acid residues were deprotonated. Similar spectral changes appeared when ADP was released from its precursor. The release of P i from the same caged molecule did not induce similar changes. Changes in tertiary structure occurring during the binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total reflectance FTIR spectroscopy (ATR-FTIR). At least 129 amide protons were involved in a tertiary structure change induced by ATP. This suggested that secondary structure change transduced a much larger tertiary structure modification.Keywords: H 1 /K 1 -ATPase; caged-compounds; catalytic cycle; ATR-FTIR spectroscopy.The gastric H 1 /K 1 -ATPase is the electroneutral ion pump responsible for acid secretion in the gastric mucosa. In this process, cytosolic H 1 is exchanged for extracellular K 1 . The gastric H 1 /K 1 -ATPase is a member of the P-ATPase family and shares many features, including sequence homologies and enzymatic mechanisms with other members such as the Ca 21 -ATPase [1], and especially the Na 1 /K 1 -ATPase [2]. Enzymes of this large family are characterized by the formation of a covalent aspartylphosphate group during their catalytic cycle and the transition between two major conformations called E1 and E2.From a structural point of view, this enzyme is composed of a catalytic a subunit (114 kDa) and a glycosylated b subunit (33 kDa). The a subunit, a membrane multispanning polypeptide, is responsible for the coupling of ATP hydrolysis with ion transport across the membrane. The b subunit, a single membrane spanning peptide, is required for the proper assembly and targeting of the entire protein. The a subunit of the H 1 /K 1 -ATPase consists of 1034 amino-acid residues [2]. It contains the phosphorylation site (Asp385) and Lys517, which is preferentially labeled by fluorescein 5 H -isothiocyanate (FITC). The models based on hydrophobicity profiles and different experimental results predict 10 membrane spanning segments for the a subunit. Although segments M9 and M10 have never been isolated after proteolysis, genetics experiments support this view [2,3]; it is also consistent with the recently published structure of the Ca 21 -ATPase [4]. The b subunit consists of 290 amino-acid residu...

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Structural difference in the H⁺,K⁺‐ATPase between the E1 and E2 conformations

Cited by 8 publications

References 52 publications

Formation of Protein–Birnessite Complex: XRD, FTIR, and AFM Analysis

Formation of Protein–Birnessite Complex: XRD, FTIR, and AFM Analysis

Difference between the E1 and E2 conformations of gastric H⁺/K⁺‐ATPase in a multilamellar lipid film system

Monitoring of secondary and tertiary structure changes in the gastric H⁺/K⁺‐ATPase by infrared spectroscopy

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Structural difference in the H+,K+‐ATPase between the E1 and E2 conformations

Cited by 8 publications

References 52 publications

Formation of Protein–Birnessite Complex: XRD, FTIR, and AFM Analysis

Formation of Protein–Birnessite Complex: XRD, FTIR, and AFM Analysis

Difference between the E1 and E2 conformations of gastric H+/K+‐ATPase in a multilamellar lipid film system

Monitoring of secondary and tertiary structure changes in the gastric H+/K+‐ATPase by infrared spectroscopy

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Structural difference in the H⁺,K⁺‐ATPase between the E1 and E2 conformations

Difference between the E1 and E2 conformations of gastric H⁺/K⁺‐ATPase in a multilamellar lipid film system

Monitoring of secondary and tertiary structure changes in the gastric H⁺/K⁺‐ATPase by infrared spectroscopy