Modulating Liposome Surface Charge for Maximized ATP Regeneration in Synthetic Nanovesicles

Overview

Journal ACS Synth Biol

Publisher American Chemical Society

Specialties Biotechnology
Molecular Biology

Date 2024 Nov 26

PMID 39592139

Authors

Sabina Deutschmann

Stefan Theodore Tauber

Lukas Rimle

Olivier Biner

Martin Schori

Ana-Marija Stanic

Christoph von Ballmoos

Affiliations

Soon will be listed here.

Abstract

In vitro reconstructed minimal respiratory chains are powerful tools to investigate molecular interactions between the different enzyme components and how they are influenced by their environment. One such system is the coreconstitution of the terminal cytochrome oxidase and the ATP synthase from into liposomes, where the ATP synthase activity is driven through a proton motive force () created by the oxidase. The proton pumping activity of the oxidase is initiated using the artificial electron mediator short-chain ubiquinone and electron source DTT. Here, we extend this system and use either complex II or NDH-2 and succinate or NADH, respectively, as electron entry points employing the natural long-chain ubiquinone Q or Q. By testing different lipid compositions, we identify that negatively charged lipids are a prerequisite to allow effective NDH-2 activity. Simultaneously, negatively charged lipids decrease the overall formation and ATP synthesis rates. We find that orientation of the oxidase in liposomal membranes is governed by electrostatic interactions between enzyme and membrane surface, where positively charged lipids yield the desired oxidase orientation but hinder reduction of the quinone pool by NDH-2. To overcome this conundrum, we exploit ionizable lipids, which are either neutral or positively charged depending on the pH value. We first coreconstituted oxidase and ATP synthase into temporarily positively charged liposomes, followed by fusion with negatively charged empty liposomes at low pH. An increase of the pH to physiological values renders these proteoliposomes overall negatively charged, making them compatible with quinone reduction via NDH-2. Using this strategy, we not only succeeded in orienting the oxidase essentially unidirectionally into liposomes but also found up to 3-fold increased ATP synthesis rates through the usage of natural, long-chain quinones in combination with the substrate NADH compared to the synthetic electron donor/mediator pair.

References

Paradies G, Paradies V, De Benedictis V, Ruggiero F, Petrosillo G . Functional role of cardiolipin in mitochondrial bioenergetics. Biochim Biophys Acta. 2013; 1837(4):408-17. DOI: 10.1016/j.bbabio.2013.10.006. View

Heikal A, Nakatani Y, Dunn E, Weimar M, Day C, Baker E . Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation. Mol Microbiol. 2014; 91(5):950-64. DOI: 10.1111/mmi.12507. View

Sanden T, Salomonsson L, Brzezinski P, Widengren J . Surface-coupled proton exchange of a membrane-bound proton acceptor. Proc Natl Acad Sci U S A. 2010; 107(9):4129-34. PMC: 2840142. DOI: 10.1073/pnas.0908671107. View

Ishmukhametov R, Russell A, Berry R . A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes. Nat Commun. 2016; 7:13025. PMC: 5059690. DOI: 10.1038/ncomms13025. View

Biner O, G Fedor J, Yin Z, Hirst J . Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration. ACS Synth Biol. 2020; 9(6):1450-1459. PMC: 7611821. DOI: 10.1021/acssynbio.0c00110. View

Adelroth P, Brzezinski P . Surface-mediated proton-transfer reactions in membrane-bound proteins. Biochim Biophys Acta. 2004; 1655(1-3):102-15. DOI: 10.1016/j.bbabio.2003.10.018. View

von Ballmoos C, Biner O, Nilsson T, Brzezinski P . Mimicking respiratory phosphorylation using purified enzymes. Biochim Biophys Acta. 2015; 1857(4):321-31. DOI: 10.1016/j.bbabio.2015.12.007. View

Ritzmann N, Thoma J, Hirschi S, Kalbermatter D, Fotiadis D, Muller D . Fusion Domains Guide the Oriented Insertion of Light-Driven Proton Pumps into Liposomes. Biophys J. 2017; 113(6):1181-1186. PMC: 5607040. DOI: 10.1016/j.bpj.2017.06.022. View

Amati A, Graf S, Deutschmann S, Dolder N, von Ballmoos C . Current problems and future avenues in proteoliposome research. Biochem Soc Trans. 2020; 48(4):1473-1492. DOI: 10.1042/BST20190966. View

10.

Biner O, Schick T, Muller Y, von Ballmoos C . Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion. FEBS Lett. 2016; 590(14):2051-62. DOI: 10.1002/1873-3468.12233. View

11.

von Heijne G . Membrane-protein topology. Nat Rev Mol Cell Biol. 2006; 7(12):909-18. DOI: 10.1038/nrm2063. View

12.

Gao Y, Zhang Y, Hakke S, Mohren R, Sijbers L, Peters P . Cryo-EM structure of cytochrome bo quinol oxidase assembled in peptidiscs reveals an "open" conformation for potential ubiquinone-8 release. Biochim Biophys Acta Bioenerg. 2024; 1865(3):149045. DOI: 10.1016/j.bbabio.2024.149045. View

13.

Sikkema H, Gaastra B, Pols T, Poolman B . Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells. Chembiochem. 2019; 20(20):2581-2592. DOI: 10.1002/cbic.201900398. View

14.

Smondyrev A, Voth G . Molecular dynamics simulation of proton transport near the surface of a phospholipid membrane. Biophys J. 2002; 82(3):1460-8. PMC: 1301947. DOI: 10.1016/S0006-3495(02)75500-8. View

15.

Toth A, Meyrat A, Stoldt S, Santiago R, Wenzel D, Jakobs S . Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes. Proc Natl Acad Sci U S A. 2020; 117(5):2412-2421. PMC: 7007565. DOI: 10.1073/pnas.1917968117. View

16.

Pols T, Sikkema H, Gaastra B, Frallicciardi J, Smigiel W, Singh S . A synthetic metabolic network for physicochemical homeostasis. Nat Commun. 2019; 10(1):4239. PMC: 6751199. DOI: 10.1038/s41467-019-12287-2. View

17.

Rumbley J, Furlong Nickels E, Gennis R . One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase. Biochim Biophys Acta. 1997; 1340(1):131-42. DOI: 10.1016/s0167-4838(97)00036-8. View

18.

Warren G, Toon P, Birdsall N, Lee A, Metcalfe J . Reconstitution of a calcium pump using defined membrane components. Proc Natl Acad Sci U S A. 1974; 71(3):622-6. PMC: 388063. DOI: 10.1073/pnas.71.3.622. View

19.

von Heijne G, Gavel Y . Topogenic signals in integral membrane proteins. Eur J Biochem. 1988; 174(4):671-8. DOI: 10.1111/j.1432-1033.1988.tb14150.x. View

20.

Veit S, Paweletz L, Bohr S, Menon A, Hatzakis N, Pomorski T . Single Vesicle Fluorescence-Bleaching Assay for Multi-Parameter Analysis of Proteoliposomes by Total Internal Reflection Fluorescence Microscopy. ACS Appl Mater Interfaces. 2022; 14(26):29659-29667. PMC: 11194769. DOI: 10.1021/acsami.2c07454. View