Name: single liposome measurements for the study of proton-pumping membrane enzymes using electrochemistry and fluorescent microscopy
Uploaded: 2019-02-21T13:00:00
Description: Watch this Scientific Journal Video about Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy at JoVE.com

The authors acknowledge the BBSRC (BB/P005454/1) for financial support. NH was funded by the VILLUM Foundation Young Investigator Program.

1. Preparation of a Lipid/UQ-10/FDLL Mix
NOTE: E. coli lipids used for the liposomes&#8217; preparation should be aliquoted and thoroughly mixed with ubiquinone-10 (enzyme substrate) and long-wavelength fluorescent dye-labeled lipids (for liposomes size determination) prior to the reconstitution.
<ol>
	<li>Using a glass syringe, transfer 200 &#181;L of chloroform stock of lipid polar extract from Escherichia coli (25 mg/mL) into glass vials to make 5 mg aliquots.</...

<ol>
	<li>Claessen, V. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Biomolecule+Kinetics:+The+Art+of+Studying+a+Single+Enzyme.">Single-Biomolecule Kinetics: The Art of Studying a Single Enzyme.</a> Annual Review of Analytical Chemistry. 3 (1), 319-340 (2010).</li><li>Rojek, M. J., Walt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+Single+Enzyme+Molecules+Interconvert+between+Activity+States+upon+Heating.">Observing Single Enzyme Molecules Interconvert between Activity States upon Heating.</a> PLoS ONE. 9 (1), e86224 (2014).</li><li>Velonia, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Enzyme+Kinetics+of+CALB-Catalyzed+Hydrolysis.">Single-Enzyme Kinetics of CALB-Catalyzed Hydrolysis.</a> Angewandte Chemie International Edition. 44 (4), 560-564 (2005).</li><li>Lu, H. P., Xun, L., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-molecule enzymatic dynamics. 282 (5395), 1877-1882 (1998).</li><li>Engelkamp, H., Hatzakis, N. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+separation+in+mixed+self-assembled+monolayers+and+its+effect+on+biomimetic+membranes.">Phase separation in mixed self-assembled monolayers and its effect on biomimetic membranes.</a> Sensors and Actuators, B: Chemical. 124 (2), 501-509 (2007).</li><li>Barsoukov, E., Macdonald, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Impedance Spectroscopy Theory, Experiment, and Applications. , (2005).</li><li>Jeuken, L. J. C., Connell, S. D., Henderson, P. J. F., Gennis, R. B., Evans, S. D., Bushby, R. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Timelapse (movie) of single vesicles fluorescence change upon potential application. , (2018).</li><li>Li, M., Khan, S., Rong, H., Tuma, R., Hatzakis, N. S., Jeuken, L. J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+membrane+curvature+and+pH+on+proton+pumping+activity+of+single+cytochrome+bo3enzymes.">Effects of membrane curvature and pH on proton pumping activity of single cytochrome bo3enzymes.</a> Biochimica et Biophysica Acta - Bioenergetics. 1858 (9), 763-770 (2017).</li><li>Li, M., Tuma, R., Jeuken, L. J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MATLAB code for the analysis of proton transport activity in single liposomes. , (2017).</li><li>Li, M., Tuma, R., Jeuken, L. J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Time-resolved fluorescence microscopic data (traces) of individual lipid vesicles with proton transport activity. , (2017).</li><li>Paula, S., Volkov, A. G., Van Hoek, A. N., Haines, T. H., Deamer, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeation+of+protons,+potassium+ions,+and+small+polar+molecules+through+phospholipid+bilayers+as+a+function+of+membrane+thickness.">Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness.</a> Biophysical Journal. 70 (1), 339-348 (1996).</li><li>Brookes, P. S., Hulbert, A. J., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proton+permeability+of+liposomes+made+from+mitochondria)+inner+membrane+phospholipids:+No+effect+of+fatty+acid+composition.">The proton permeability of liposomes made from mitochondria) inner membrane phospholipids: No effect of fatty acid composition.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1330 (2), 157-164 (1997).</li><li>Eriksson, E. K., Agmo Hern&#225;ndez, V., Edwards, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ubiquinone-10+on+the+stability+of+biomimetic+membranes+of+relevance+for+the+inner+mitochondrial+membrane.">Effect of ubiquinone-10 on the stability of biomimetic membranes of relevance for the inner mitochondrial membrane.</a> Biochimica et Biophysica Acta - Biomembranes. 1860 (5), 1205-1215 (2018).</li><li>Seneviratne, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reconstitution+method+for+integral+membrane+proteins+in+hybrid+lipid-polymer+vesicles+for+enhanced+functional+durability.">A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability.</a> Methods. , 1-8 (2018).</li><li>Veshaguri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+proton+pumping+by+a+eukaryote+P-type+ATPase.">Direct observation of proton pumping by a eukaryote P-type ATPase.</a> Science. 351 (6280), 1-6 (2016).</li></ol>

The method described is suitable to study proton pumping by respiratory membrane proteins that can be reconstituted into liposomes and are able to exchange electrons with the quinone pool. Proton pumping activity can be monitored at the single-enzyme level using pH-sensitive (ratiometric) dyes encapsulated in the liposome lumen (Figure 1A).
The method relies on the ability of ubiquinone (or other quinones), incorporated into the lipid bilayer, to exchange electron...

Information about enzyme mechanisms and kinetics is usually obtained on the ensemble or macroscale level with enzyme population in the thousands to millions of molecules, where measurements represent a statistical average. It is known, however, that complex macromolecules such as enzymes may demonstrate heterogeneity in their behavior and molecular mechanisms observed at the ensemble level are not necessarily valid for every molecule. Such deviations on the individual molecule scale have been extensively confirmed by studies of single enzymes with a variety of methods emerging during the last two decades1. Notably, fluorescence detection of individual enzyme activity has been used to investigate heterogeneity of enzymes activity2,3 or discover the so-called memory effect (periods of high enzymes activity succeeded by periods of low activity and vice versa)4,5.
Many single enzyme studies require that the enzymes are immobilized on the surface or spatially fixed in another way to remain sufficiently long in the field of view for continuous observation. Enzyme encapsulation into liposomes has been shown to enable enzyme immobilization while preventing any negative impact due the surface-enzyme or protein-protein interactions6,7. In addition, liposomes offer a unique possibility to study single membrane proteins in their natural lipid bilayer environment8,9,10.
A class of membrane proteins, transporters, exercises a directional translocation of substances across the cell membrane, a behavior that can only be studied when proteins are reconstituted into the lipid bilayers (e.g., liposomes)11,12,13. For example, proton translocation, exhibited by several enzymes of prokaryotic and eukaryotic electron transport chains, plays an important role in cellular respiration by creating a proton-motive force used for ATP synthesis. In this case, the proton pumping activity is coupled to the electron transfer, although the detailed mechanism of this process often remains elusive.
Recently, we demonstrated the possibility to couple fluorescent detection with electrochemistry to study proton pumping activity of single enzymes of the terminal ubiquinol oxidase of Escherichia coli (cytochrome bo3) reconstituted in the liposomes14. This was achieved by encapsulation of a pH-sensitive membrane-impermeable fluorescent dye into the lumen of liposomes prepared from E. coli polar lipids (Figure 1A). The protein amount was optimized so that most liposomes either contained no or only one reconstituted enzyme molecule (according to Poisson distribution). The two substrates of cytochrome bo3 were provided by adding ubiquinone to the lipid mix that formed the liposomes and (ambient) oxygen in solution. The liposomes are then sparsely adsorbed on a semi-transparent ultra-smooth gold electrode, covered with a self-assembled monolayer of 6-mercaptohexanol. Finally, the electrode is mounted on the bottom of a simple spectroelectrochemical cell (Figure 1B). Electrochemical control of the quinone pool redox state allows one to flexibly trigger or to stop the enzymatic reaction at any moment, while the pH-sensitive dye is used to monitor pH changes inside the lumen of the liposomes as a result of proton translocation by the enzymes. By using the fluorescence intensity of a second, lipid-bound fluorescent dye, the size and volume of individual liposomes can be determined and thus the quantification of enzyme proton pumping activity. Using this technique, we notably found that cytochrome bo3 molecules are able to enter into a spontaneous leak state that rapidly dissipates the proton motive force. The goal of this article is to introduce the technique of single liposome measurements in detail.

<table>
	<tbody>
		<tr>
			<td>6-Mercapto-1-hexanol (6MH)</td>
			<td>Sigma</td>
			<td>451088</td>
			<td>97%</td>
		</tr>
		<tr>
			<td>8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS)</td>
			<td>BioChemika</td>
			<td>56360</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium holder (Electrochemical cell)</td>
			<td>Custom-made</td>
			<td></td>
			<td>30x30x7 mm; inner diameter: 26 mm; hole diameter: 15 mm</td>
		</tr>
		<tr>
			<td>Auxiliary electrode</td>
			<td></td>
			<td></td>
			<td>platinum wire</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR Chemicals</td>
			<td>83627</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli polar lipids</td>
			<td>Avanti</td>
			<td>100600C</td>
			<td>25 mg/mL in chloroform</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>EPO-TEK</td>
			<td>301-2FL</td>
			<td>low fluorescence epoxy</td>
		</tr>
		<tr>
			<td>Fiji (ImageJ 1.52d)</td>
			<td></td>
			<td></td>
			<td>Required plugins: StackReg and TurboReg (http://bigwww.epfl.ch/thevenaz/stackreg/)</td>
		</tr>
		<tr>
			<td>Filter cube (&#34;ATTO633&#34;)</td>
			<td>Chroma Technology Corporation</td>
			<td></td>
			<td>Ex: 620/60 nm; DM: 660 nm; Em: 700/75 nm</td>
		</tr>
		<tr>
			<td>Filter cube (&#34;HPTS1&#34;)</td>
			<td>Chroma Technology Corporation</td>
			<td></td>
			<td>Ex: 470/20 nm; DM: 500 nm; Em: 535/48 nm</td>
		</tr>
		<tr>
			<td>Filter cube (&#34;HPTS2&#34;)</td>
			<td>Chroma Technology Corporation</td>
			<td></td>
			<td>Ex: 410/300 nm; DM: 500 nm; Em: 535/48 nm</td>
		</tr>
		<tr>
			<td>Filter cube (&#34;Texas Red&#34;)</td>
			<td>Chroma Technology Corporation</td>
			<td></td>
			<td>Ex: 560/55 nm; DM: 595 nm; Em: 645/75 nm</td>
		</tr>
		<tr>
			<td>Fluorescent dye-labelled lipids (FDLL)</td>
			<td>ThermoFisher Scientific</td>
			<td>T1395MP</td>
			<td>TexasRed-DHPC was used in this work (&#955;exc 595 nm; &#955;em 615 nm)</td>
		</tr>
		<tr>
			<td>Fluorescent dye-labelled lipids (FDLL) (alternative)</td>
			<td>ATTO-TEC</td>
			<td>AD 633-161</td>
			<td>ATTO633-DOPE can be used as alternative (&#955;exc 630 nm; &#955;em 651 nm)</td>
		</tr>
		<tr>
			<td>Gel filtration column</td>
			<td>GE Healthcare</td>
			<td>28-9893-33</td>
			<td>HiLoad 16/600 Superdex 75 pg, for additional protein purification</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>VWR International</td>
			<td>631-0172</td>
			<td>No 1.5</td>
		</tr>
		<tr>
			<td>Glass syringe</td>
			<td>Hamilton</td>
			<td>1725 RNR</td>
			<td>250 &#181;L</td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Scientific Glass Laboratories Ltd</td>
			<td>T101/V1</td>
			<td>1.75 mL capacity</td>
		</tr>
		<tr>
			<td>Gold</td>
			<td>GoodFellow</td>
			<td></td>
			<td>99.99%</td>
		</tr>
		<tr>
			<td>Gramacidin</td>
			<td>Sigma</td>
			<td>G5002</td>
			<td></td>
		</tr>
		<tr>
			<td>Mercury sulfate reference electrode</td>
			<td>Radiometer (Hash)</td>
			<td>E21M012</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>Minispin NL040</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Andor</td>
			<td>Zyla 5.5 sCMOS</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Lamp</td>
			<td>Nikon</td>
			<td>Intensilight C-HGFI</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR 5.0.2</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope acquisition software</td>
		</tr>
		<tr>
			<td>n-Octyl &#946;-D-glucopyranoside</td>
			<td>Melford Laboratories</td>
			<td>B2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Nova 1.10</td>
			<td>Metrohm</td>
			<td></td>
			<td>Potentiostat control software</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Nikon</td>
			<td>Plan Apo &#955; 60x/1.4 oil</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 2017</td>
			<td>OriginLab</td>
			<td></td>
			<td>Plotting software</td>
		</tr>
		<tr>
			<td>O-ring (Electrochemical cell)</td>
			<td>Orinoko</td>
			<td></td>
			<td>Inner diameter: 16 mm; cross section: 1.5 mm</td>
		</tr>
		<tr>
			<td>Plastic tubes</td>
			<td>Eppendorf</td>
			<td>3810X</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Polystyrene microbeads</td>
			<td>Bio-RAD</td>
			<td>152-3920</td>
			<td>Biobeads, 20-50 mesh</td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>Metrohm Autolab</td>
			<td>PGSTAT 128N</td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>CH Instruments</td>
			<td>CHI604C</td>
			<td></td>
		</tr>
		<tr>
			<td>Scripting software</td>
			<td>Matlab</td>
			<td>R2017a</td>
			<td>Required toolboxes: &#39;Image Processing Toolbox&#39;, &#39;Parallel Computing Toolbox&#39;, &#39;Curve Fitting Toolbox&#39;, &#39;System Identification Toolbox&#39;, &#39;Optimization Toolbox&#39;</td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>IDB Technologies LTD</td>
			<td>Si-C2 (N&#60;100&#62;P)</td>
			<td>&#216; 25 mm, 525 um thick</td>
		</tr>
		<tr>
			<td>Teflon cell (Electrochemical cell)</td>
			<td>Custom-made</td>
			<td></td>
			<td>Outer diameter: 26 mm; inner diameter: 13.5 mm</td>
		</tr>
		<tr>
			<td>Temple-Stripped Ultra-Flat Gold Surfaces</td>
			<td>Platypus Technologies</td>
			<td>AU.1000.SWTSG</td>
			<td>Alternative ready-to-use ultra-flat gold surfaces (Thickness below 100 nm on demand)</td>
		</tr>
		<tr>
			<td>Thin micropipette tips</td>
			<td>Sarstedt</td>
			<td>70.1190.100</td>
			<td>or similar gelloader tips 200 &#181;L</td>
		</tr>
		<tr>
			<td>Ubiquinone-10</td>
			<td>Sigma</td>
			<td>C-9538</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td>L-80XP</td>
			<td>with Ti 45 rotor</td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Fisher Scientific</td>
			<td>FB15063</td>
			<td></td>
		</tr>
	</tbody>
</table>

single liposome measurements for the study of proton-pumping membrane enzymes using electrochemistry and fluorescent microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

The quality of the gold-modified cover slip (the electrode) with a SAM of 6MH is checked before each experiment with electrochemical impedance spectroscopy. Figure 2A shows representative Cole-Cole plots measured using electrochemical impedance spectroscopy before and after liposomes are adsorbed. If the quality of SAM is sufficient, impedance spectroscopy should demonstrate an almost pure capacitive behavior resulting in a semi-circle Cole-Cole plot. The dia...

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Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

This method allows the study of proton transport activity of respiratory membrane enzymes like cytochrome BO3 in a natural lipid bi-layer environment. The main advantages of this single enzyme technique is the possibility to start and stop enzymatic reactions at any moment using electrochemistry. Using a glass syringe transfer 200 microliters of chloroform stock of lipid polar extract from Escherichia coli into glass vials to make five milligram aliquots.Add 50 microliters of one milligram per milliliter ubiquinone 10 to the lipids to make the final ratio of one to 100 ubiquinone 10 to lipids. To the mixture add 20 microliters of one milligram per milliliter long wave length fluorescent dye labeled lipid abbreviated FDLL. Homogenize the chloroform solution by short vortexing and evaporate most of the chloroform under a gentle nitrogen or argon flow.Remove the chloroform traces entirely by further evaporation under vacuum for at least one hour. Add 312.5 microliters of 40 millimolar MOPS pH 7.4 buffer to one aliquot of lipids ubiquinone 10 FDLL dry mix. Mix the vortex til the lipid film is fully re-suspended followed by two minutes of treatment in an ultrasonic bath.Next add 125 microliters of 25 millimolar HPTS, a pH sensitive fluorescent dye that will be encapsulated inside the liposomes. Now add 137.5 microliters of 250 millimolar OGP surfactant. After mixing using vortex, sonicate the mixture in an ultrasonic water bath for 10 minutes to ensure all lipids are solubilized into surfactant micelles.Then transfer the dispersion into a 1.5 milliliter plastic tube. Add the required amount of cytochrome BO3 and add ultra pure water to make a total volume of 50 microliters. Incubate the reconstitution mixture at four degrees celsius for 10 minutes on a roller mixer.Next weigh 50 milligrams of polystyrene microbeads into each of the two 1.5 milliliter tube caps. Then weigh 100 milligrams of polystyrene microbeads into another two 1.5 milliliter tube caps. Close the caps with paraffin film to prevent drying.Add the first 50 milligrams of polystyrene microbeads into the reconstitution mixture by putting the cap with polystyrene microbeads on the 1.5 milliliter plastic tube with the prepared dispersion. Then perform a short spin for a couple of seconds to bring microbeads to the bottom of the tube. Incubate the suspension at four degrees celsius on a roller mixer to allow the polystyrene microbeads to absorb the surfactant for 30 minutes.Repeat the additions of polystyrene microbeads and incubations as follows. Add 50 milligram of microbeads for 60 minutes of incubation. Then add 100 milligram of microbeads for 60 minutes of incubation.And finally, add 100 milligram of microbeads for 120 minutes of incubation. Separate the proteoliposome solution from the polystyrene microbeads using a micropipette with a thin tip. Dilute the dispersion in 90 milliliters of MOPS buffer in TI 45 ultracentrifuge tube.Ultracentrifuge the dispersion using a type 45 TI rotor at 125, 000 times G for one hour to pellet the proteoliposomes. Following ultracentrifugation, discard the supernatant and rinse the pellets with MOPS buffer without resuspending the pellet. After discarding the rinsing buffer, re-suspend the proteoliposomes in 500 microliters of MOPS buffer by pipetting it back and forth with a thin tipped micropipette before transferring the suspension to a 1.5 milliliter plastic tube.Following centrifugation for five minutes at 12, 000 times G to remove the debris, transfer the supernatant which is the reconstituted proteoliposomes into a new vial. Glue up to nine glass cover slips onto an evaporated gold surface using bi-component low-fluorescence epoxy. Cure the glue at 80 degrees celsius for four hours.Just before modification with a self-assembled mono-layer, detach the glass cover slips from the silicone wafers with a blade. Due to the thinness of the cover slips, take care when detaching the cover slips to not crack or break the glass slides. Dip the freshly detached gold-coated cover slips into a 6-marcaptohexanol solution and leave at 20 to 25 degrees celsius overnight to form the self assembled mono-layer.The next day, remove the gold-coated cover slips from the solution and wash briefly with water or methanol and then with isopropanol. Dry the gold-coated cover slips under a gentle gas flow. Assemble the gold-coated cover slip in a spectro-electrochemical cell as diagrammed in the text protocol.Make contact to the gold with a flat wire outside an area defined by a rubber O-ring and tightly screw down the electrochemical cell on the top of it. Add two milliliters of the electrolyte buffer solution and place the reference and auxiliary electrodes in the cell. Proceed to check the quality of the self-assembled mono-layer and run blanks as described in the text protocol.Add proteoliposomes to the electrochemical cell at 0.5 milligrams per milliliter final lipids concentration and mix slightly with a pipette. Wait 30 to 60 minutes at room temperature until the absorption of proteoliposomes on the electrode surface is finished. Wash the cell by changing the buffer solution at least 10 times, but avoid leaving the electrode surface completely dry.Now, run electrochemical impedance spectroscopy at open cell potential to confirm the self-assembled mono-layer on the gold electrode remains unchanged. Run cyclic voltammograms with scan rates 10 and 100 millivolts per second to observe catalatic ubiquinol oxidation and oxygen reduction by cytochrome BO3 at onset potentials of electrochemical quinone reduction. Modify the gold electrode as before but using five micrograms per milliliter proteoliposomes.For single enzyme studies reduce the cytochrome BO3 to lipid ratio to between 0.1 and 0.2%Place a drop of immersion oil and then the electrochemical cell on the 60 times oil objective of an inverted fluorescence microscope. Using appropriate filters for FDLL fluorescence, focus on the electrode surface. Single liposomes should appear as bright spots at the diffraction limit of the microscope objective.Take an image of FDLL florescence. Switch to the one of HPTS florescence filter sets on the microscope to verify that HTPS florescence is clearly visible and distinguishable from the background. Increase the light intensity if it is not the case.Program the microscope software to perform a timed image acquisition by alternating two HTPS filter sets. To do so, set a one second exposure then navigate to menu applications. Then Define/Run ND Acquisition and select two HTPS channels.Set the delay between image acquisitions at minimum. In this experiment use 0.3 second delay and five minute total duration. Adjust the settings of the potentiostat to change the potential during the image acquisition as indicated in the text protocol.Now, simultaneously run the timed image acquisition on the microscope and the potential sequence on the potentiostat by manually starting both measurements at the same time. Shown here are fluorescence images of liposomes absorbed on the electrodes at three different coverages. The dye containing liposomes are visible on the images as bright spots.The central part of the image was photo-bleached to reveal the background florescence level. The images on two HTPS channels are super imposable where the ratio between the two channels corresponds to a pH of 7.4 used in this experiment. A larger number of liposomes are visible with the FDLL channel, which indicates the presence of liposomes that have no HPTS encapsulated.The difference between the HPTS and FDLL channels is more pronounced at higher coverages. Possibly because at high liposome coverage, liposomes are more likely to burst or fuse on the surface. Here, the change of a liposome florescence on two HTPS channels is shown as a 3D surface plot of the corresponding area during 300 seconds of the experiment.The reductive potential was applied between 60 seconds and 180 seconds. The corresponding plot of volume metric intensity ratio of two HPTS channels versus time is shown. Shown here are the medians of all vesicle pH changes within a single image when cytochrome BO3 content is 1.3%An increase in pH is clearly visible when a potential between 0.1 and 0.3 volts is applied.When cytochrome BO3 content is a much lower value of 0.1%the median curves become almost indistinguishable from those of empty liposomes. It is important to ensure complete removal of the surfactant from the liposome dispensations since the residuals may increase the permeability of the liposomes to protons. These methods allow us to ask specific questions on the natural lipophilic quinone substrate in contrast to the often used water-soluble quinone analogues.Further more, the single enzyme experiments identified a previously unknown leak state where the protein acts as a proton channel where protons freely flow backwards.

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