Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes

Abstract

The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min-' mg ATP synthase-was acheved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-' mg-' found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches.The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches. The final activities depended on both the purification method for the two proteins and the coreconstitution procedure. Furthermore, the ratio (lipid to bacteriorhodopsin to ATP synthase) could be optimized. Light-driven ATP synthesis depends also on the type of detergent used. The best result was obtained by deoxycholate. Also the relationship between proton translocation (by bacteriorhodopsin) and ATP synthesis activity was measured. A constant H+/ATP ratio was found at higher light intensities. This ratio increased strongly at lower light intensities.According to the chemiosmotic theory [l] ATP synthesis in chloroplasts, bacteria and mitochondria, catalyzed by ATP synthase, is driven by an electrochemical gradient of protons across the membrane. Quantitative investigations of the mechanisms of ATP synthesis in vivo are problematical owing to the complex structure of the natural systems. A way to overcome some difficulties is the coreconstitution of an isolated proton pump and a purified ATP synthase. In principle the function of such systems has been demonstrated [2-lo]. The proton pump commonly used in these investigations was bacteriorhodopsin in the form of purple membrane patches isolated from Halobacterium halobium. The most effective coreconstitution system yields ATP synthesis rates up to 10% of the maximal in vivo rate when yeast mitochondria1 ATP synthase is used [4, 81. Unfortunately the reported rates for liposomes with incorporated bacterial ATP synthases are less than 1 YO of the in vivo rate [3, 51. This might be due to the instability of the delipidated enzyme and/or the heterogeneous distribution of the ATP synthase and the bacteriorhodopsin among the liposomes. The ATP synthase complex from Rhodospirillum rubrum has been chosen because of the higher stability of the FoFl complex compared with that of other bacterial ATP synthases (Escherichia coli, Micrococcus luteus). In this paper we show that monomeric bacteriorhodopsin, delipidated by a HPLC procedure [l 1 -131, is superior to purple membrane patches in coreconstitution experiments. Various coreconstitution procedures have been optimized. The protein distribution among the liposomes has been analyzed by density gradient centrifugation. MATERIALS AND METHODSThe detergents deoxycholate, taurodeoxycholate, and 3-[(3-cholamidopropyl)dimethylammonio]-propane sulphonate (Chaps) were purchased from Sigma (Miinchen). Luciferin, luciferase, ATP, and ADP (K salt) were product...

Light‐Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles

“…We decided for a Triton‐X‐100 mediated reconstitution, because octylglucoside has been reported to inactivate MF O F 1 ‐and CF O F 1 ‐ATP synthase and also led to much lower ATP production rates in our experiments (data not shown). For coupling bR with ATP synthase in lipid vesicles, different bR/F O F 1 ‐ATP synthase ratios, ranging from 1 : 1 to 170 : 1, have been covered. The respective ATP production rates are increasing with the amount of bR (Figure S2), presumably caused by the establishment of higher driving forces.…”

Artificial Organelles for Energy Regeneration

“…Another critical point for the performance of these coassemblies is the bRH/ATP synthase ratio per liposome. Different bRH/ATP synthase ratios, ranging from 1/1 to 170/1, have been reported. To characterize the performance of the light‐driven energy conversion, different methods to analyze the activity of both membrane‐inserted building blocks were developed.…”

Multistimuli Sensing Adhesion Unit for the Self‐Positioning of Minimal Synthetic Cells

“…We co‐reconstituted EF o F 1 ATP synthase (Figure S6, Supporting Information) from E. coli and bR (Figure S7, Supporting Information) from H. salinarium in phosphatidylcholine (soy PC) liposomes that were supplemented with lipids baring Ni 2+ ‐NTA head groups (10 mol%, Ni 2+ ‐NTA‐DGS), yielding vesicles with a mean diameter of 142 nm ( Figure a and Figure S8, Supporting Information). [ 27,35,36 ] For the ATP production, the orientation of the transmembrane proteins, bR and ATP synthase, in the lipid membrane are important and ATP will only be produced if the majority is in the correct orientation. In the here used method, the ATP synthase is mainly orientated with the hydrophilic head outward and the hydrophilic head is unlikely to go through the hydrophobic membrane during reconstitution, resulting in the active orrientation.…”