ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin: 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin

Bruno Pitard, Peter Richard, Mireia Duñach, Guy Girault, Jean Louis Rigaud

Research output: Contribution to journalArticleScientificpeer-review

46 Citations (Scopus)

Abstract

Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40°C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitution were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TF0F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP · min-1 · mg TF0F1 -1 in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis by TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.

Original languageEnglish
Pages (from-to)769-778
Number of pages10
JournalEuropean Journal of Biochemistry
Volume235
Issue number3
DOIs
Publication statusPublished - 1 Jan 1996
MoE publication typeNot Eligible

Fingerprint

Bacteriorhodopsins
Bacilli
Liposomes
Bacillus
Adenosine Triphosphate
Detergents
Octoxynol
Phospholipids
Light
Proteins
Mitochondrial Proton-Translocating ATPases
Cholesterol
Sodium Cholate
Lipids
Chemical activation
Phosphatidic Acids
Glycols
Deoxycholic Acid
Proton-Translocating ATPases
Sterols

Keywords

  • ATP synthesis
  • Bacteriorhodopsin
  • FF ATP synthase
  • Proteoliposomes
  • Reconstitution

Cite this

@article{9e232dd282a243e6913f532a8a4253fe,
title = "ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin: 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin",
abstract = "Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40°C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitution were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TF0F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65{\%} decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP · min-1 · mg TF0F1 -1 in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis by TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.",
keywords = "ATP synthesis, Bacteriorhodopsin, FF ATP synthase, Proteoliposomes, Reconstitution",
author = "Bruno Pitard and Peter Richard and Mireia Du{\~n}ach and Guy Girault and Rigaud, {Jean Louis}",
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ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin : 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin. / Pitard, Bruno; Richard, Peter; Duñach, Mireia; Girault, Guy; Rigaud, Jean Louis.

In: European Journal of Biochemistry, Vol. 235, No. 3, 01.01.1996, p. 769-778.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin

T2 - 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin

AU - Pitard, Bruno

AU - Richard, Peter

AU - Duñach, Mireia

AU - Girault, Guy

AU - Rigaud, Jean Louis

PY - 1996/1/1

Y1 - 1996/1/1

N2 - Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40°C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitution were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TF0F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP · min-1 · mg TF0F1 -1 in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis by TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.

AB - Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40°C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitution were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TF0F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP · min-1 · mg TF0F1 -1 in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis by TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.

KW - ATP synthesis

KW - Bacteriorhodopsin

KW - FF ATP synthase

KW - Proteoliposomes

KW - Reconstitution

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