Name: a fluorescence-based assay of phospholipid scramblase activity
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

This study was supported by the Velux Stiftung (A.K.M.), NIH grant EY024207 (A.K.M.) and the Austrian Science Fund (FWF) project J3686 (B.P.).

1. Preparation of Liposomes and Proteoliposomes
<ol>
	<li>Liposome Formation
	<ol>
		<li>Using a glass syringe, add 1,435 &#181;l POPC (25 mg/ml, in chloroform) and 160 &#181;l POPG (25 mg/ml, in chloroform) to a round bottom flask to obtain 52.5 &#181;mol lipids in a molar ratio of POPC:POPG = 9:1.</li>
		<li>Dry the lipids for 30 min using a rotary evaporator at a rotation speed of 145 rpm (no water bath is needed for this volume of solvent), then transfer the flask to a vacuum desiccator for at least 3 hr, or overni...

<ol>
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D., 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=Evidence+that+the+wzxE+gene+of+Escherichia+coli+K-12+encodes+a+protein+involved+in+the+transbilayer+movement+of+a+trisaccharide-lipid+intermediate+in+the+assembly+of+enterobacterial+common+antigen.">Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen.</a> J. Biol. Chem. 278, 16534-16542 (2003).</li><li>Suzuki, J., Imanishi, E., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+phosphatidylserine+by+Xk-related+protein+family+members+during+apoptosis.">Exposure of phosphatidylserine by Xk-related protein family members during apoptosis.</a> J. Biol. 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The scramblase activity assay enabled us originally to determine that opsin has phospholipid scramblase activity 2. The assay also allowed us to characterize opsin&#39;s scramblase activity by testing specificity (we used a variety of NBD-labeled reporter lipids such as NBD-phosphatidylethanolamine, labeled with NBD on an acyl chain as shown for NBD-PC in Figure 1A, or on the headgroup, NBD-sphingomyelin or NBD- phosphatidylserine 2), the effect of vesicle lipid composition (e.g...

The photoreceptor rhodopsin, a prototypical G protein-coupled receptor (reviewed for example in reference 1), is the first phospholipid scramblase to be identified and biochemically verified 2,3. Scramblases are phospholipid transporters that increase the intrinsically slow rate of transbilayer phospholipid movement to physiologically appropriate levels in a bidirectional, ATP-independent manner 4-6. Examples of their actions can be found in the endoplasmic reticulum and bacterial cytoplasmic membrane where constitutive scrambling is needed for membrane homeostasis and growth, as well as for a variety of glycosylation pathways 5. Regulated phospholipid scrambling is needed to expose phosphatidylserine (PS) on the surface of apoptotic cells where it acts as an &#34;eat-me&#34;-signal for macrophages 7 and provides a procoagulant surface on activated blood platelets to catalyze the production of protein factors needed for blood clotting. In photoreceptor disc membranes, rhodopsin&#39;s scrambling activity has been suggested to counteract the phospholipid imbalance between the two membrane leaflets of the bilayer that is generated by the ATP-dependent, unidirectional lipid flippase ABCA4 4,8,910-12.
Despite the physiological importance of scramblases, their identity remained elusive until rhodopsin was reported as a scramblase in photoreceptor discs 2, members of the TMEM16 protein family were identified as Ca2+-dependent scramblases needed for PS exposure at the plasma membrane (reviewed in reference 13), and the bacterial protein FtsW was proposed as a Lipid II scramblase required for peptidoglycan synthesis 14. These discoveries were based on the reconstitution of purified proteins in liposomes and demonstration of scramblase activity in the resulting proteoliposomes using the methodology described here. Other potential scramblases 15-21&#160;&#8212; the MurJ and AmJ proteins implicated in peptidoglycan biosynthesis, WzxE and related proteins implicated in scrambling O-antigen precursors, MprF protein needed to translocate aminoacylated phosphatidylglycerol across the bacterial cytoplasmic membrane, and Xkr8 family members that have been proposed to expose PS on the surface of apoptotic cells&#160;&#8212; remain to be tested biochemically. This highlights the importance of a robust assay to identify and characterize scramblase activity.
Here, we describe the reconstitution of purified opsin, the apoprotein of the photoreceptor rhodopsin, into large unilamellar vesicles (LUVs), and subsequent analysis of scramblase activity in the resulting proteoliposomes using a fluorescence-based assay. There are several well-described protocols available in the literature for the heterologous expression and purification of opsin, therefore we will not describe it in this protocol; we use the protocols described in Goren et al. 3 which yields FLAG-tagged, thermostable opsin at about 100 ng/&#181;l in 0.1% (w/v) dodecylmaltoside (DDM).
Reconstitution is achieved by treating LUVs with sufficient detergent so that they swell but do not dissolve. Under these conditions, a membrane protein&#160;&#8212; supplied in the form of protein-detergent micelles&#160;&#8212; will integrate into the liposomes and become reconstituted into the liposome membrane upon detergent removal, resulting in proteoliposomes. To reconstitute opsin (obtained as a purified protein in 0.1% (w/v) DDM), LUVs are prepared from a mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) and saturated with DDM before adding opsin and NBD-PC. The detergent is then removed by treating the sample with polystyrene beads.
The principle underlying the fluorescence-based assay is shown in Figure 1B. LUVs are symmetrically reconstituted with a trace amount of NBD-PC or other NBD-labeled fluorescent phospholipid reporter (Figure 1A). On adding dithionite, a membrane-impermeant dianion, NBD-PC molecules in the outer leaflet of the LUVs are rendered non-fluorescent as the nitro-group of NBD is reduced to a non-fluorescent amino-group. As neither NBD-PC molecules nor dithionite are able to traverse the membrane on the time-scale of the experiment (&#60;10 min), this results in 50% reduction of the fluorescent signal. However, if the liposomes are reconstituted with a scramblase, NBD-PC molecules in the inner leaflet can scramble rapidly to the outside where they are reduced. This results in the total loss of fluorescence in the ideal case (Figure 1C).
<img alt="Figure 1" src="/files/ftp_upload/54635/54635fig1.jpg" /> 
Figure 1: Schematic representation of the scramblase activity assay. The assay uses a fluorescent NBD-labeled reporter lipid; NBD-PC is shown (A). Large unilamellar vesicles are reconstituted with a trace amount of NBD-PC. Reconstitution produces symmetric vesicles, with NBD-PC distributed equally in the outer and inner leaflets. Dithionite (S2O42-) chemically reduces the nitro-group of NBD to a non-fluorescent amino-group. Treatment of protein-free liposomes with dithionite (B, top) causes a 50% reduction of fluorescence since only the NBD-PC molecules in the outer leaflet are reduced: dithionite is negatively charged and cannot cross the membrane to react with NBD-PC molecules in the inner leaflet. Dithionite treatment of opsin-containing proteoliposomes (B, bottom), i.e., scramblase-active proteoliposomes, results in 100% loss of fluorescence as opsin facilitates movement of NBD-PC between the inner and the outer leaflet. (C) shows idealized fluorescence traces obtained on treating protein-free liposomes and opsin-containing proteoliposomes with dithionite. The rate of fluorescence loss is the same in both cases indicating that the chemical reduction of NBD by dithionite is rate-limiting, and that scrambling occurs at a rate equal to or greater than the rate of the chemical reaction. Traces obtained from an actual experiment are shown in Figure 3. <a href="https://www.jove.com/files/ftp_upload/54635/54635fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The methods we describe can be used to reconstitute and assay other purified proteins, as well as mixtures of membrane proteins obtained, for example, by extracting microsomes with detergent 22.

<table border="0" cellpadding="0" cellspacing="0" width="1148">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:141px;">Avanti Polar Lipids</td>
			<td style="width:132px;">850457C</td>
			<td style="width:193px;">POPC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840457C</td>
			<td>POPG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-an-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>810130C</td>
			<td>NBD-PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
			<td>VWR Scientific</td>
			<td>EM-5330</td>
			<td>HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma</td>
			<td>S7653-1KG</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dodecyl-&#946;-d-maltoside&#160;</td>
			<td>Anatrace</td>
			<td>D310 5 GM</td>
			<td>DDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorimeter cuvettes</td>
			<td>sigma</td>
			<td>C0918-100EA</td>
			<td>cuvettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrofluorometer</td>
			<td colspan="2">Photon Technology International, Inc.</td>
			<td>fluorimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrosulfite technical grade, 85%</td>
			<td>Sigma</td>
			<td>157953-5G</td>
			<td>dithionite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 5 software</td>
			<td></td>
			<td></td>
			<td>Prism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>VWR</td>
			<td>JTX171-3</td>
			<td>Tris</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIPEX 10 mL extruder&#160;</td>
			<td>Northern Lipids, Inc.</td>
			<td></td>
			<td>Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman, Drain disc, PE, 25 mm</td>
			<td>Sigma</td>
			<td style="width:132px;">28156-243</td>
			<td>Disc support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nuclepore Track-Etched Membranes, 0.4 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110607</td>
			<td>400 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nucleopore Track-Etched Membranes, 0.2 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110606</td>
			<td>200 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate</td>
			<td>Sigma</td>
			<td>S3264-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR Culture Tubes, Disposable, Borosilicate Glass, 13x100 mm</td>
			<td>VWR Scientific</td>
			<td>47729-572</td>
			<td>glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perchloric acid</td>
			<td>Sigma</td>
			<td>30755-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Molybdate Tetrahydrate</td>
			<td>Sigma</td>
			<td>A-7302</td>
			<td>ammonium molybdate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Sodium L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25G</td>
			<td>sodium ascorbate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Beads SM2 adsorbents</td>
			<td>Bio Rad</td>
			<td>1523920</td>
			<td>polystyrene beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;2.0 mL Microtubes clear</td>
			<td>VWR Scientific</td>
			<td>10011-742</td>
			<td>Reconstitution tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reconstitution glass tube</td>
			<td>VWR Scientific</td>
			<td style="width:132px;">53283-800</td>
			<td>Reconstitution glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer&#160;</td>
			<td>Malvern&#160;</td>
			<td></td>
			<td>DLS</td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay of phospholipid scramblase activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe the reconstitution of opsin into LUVs to characterize its scramblase activity using a fluorescence-based assay. We analyze the results to place a lower limit on the rate of opsin-mediated phospholipid scrambling and to determine the oligomeric state in which opsin functionally reconstitutes into the vesicles.
To identify optimal reconstitution conditions, it is necessary to determine empirically the amount of deterge...

Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

A Fluorescence-based Assay of Phospholipid Scramblase Activity

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

The overall goal of this procedure is to reconstitute a typical G protein coupled receptor into large unilamellar vesicles, and to measure it's ability to carry out 80p independent translocation of phospholipids across a lipid bilayer using a flurorescence-based assay. This method can help answer key questions in the inter-cellular lipid transport field, such as what is the molecular identity of proteins that can scramble off liplipids and what is their mechanism. The main advantage of this technique is that it is very reliable and robust.Once your reconstitution procedure is established for a given lipid composition and a preferred detergent. To begin this procedure, use a glass syringe to add 1435 microliters of a 25 milligram per milliliter solution of POPC and chloroform to a round bottom flask. Add 160 microliters of a 25 milligram per milliliter solution of POPG and chloroform to obtain 52.5 micro-malipids in a nine to one molar ratio of POPC to POPG.Next, dry the lipids for 30 minutes using a rotary evaporator set to 145 RPM. Once drying is complete, transfer the flask to a vacuum desiccator for three hours at room temperature. Then, add 10 milliliters of buffer A to hydrate the died lipid film.Gently swirl the flask until a homogeneous turbid suspension is formed. Sonicate the suspension at a frequency of 40 kilohertz in a room temperature water bath for 10 minutes. Then, using an extruder, pass the sample through a membrane with a 400 nanometer pore size, 10 times.Follow this with a second cycle of extrusion, passing the sample through a membrane with a 200 nanometer pore size, four times. Set aside the resulting visibly clearer suspension, which contains large unilamellar vesicles of phospolipids about 175 nanometers in diameter. After the extrusion is complete, prepare standards in 13 by 100 millimeter glass tubes as outlined in the text protocol.Then, add 10 microliters of each LUV and proteoliposome sample to a unique glass tube and dilute with 40 microliters of deionized distilled water. Add 300 microliters of perchloric acid to each tube, and heat for one hour at 145 degrees Celsius in a heating block. Place a marble on each tube to prevent evaporation.Then let the tubes cool to room temperature and add one milliliter of deionized distilled water to each. Add 400 microliters of a freshly prepared solution containing 12 grams per liter of ammonium molybdate and 50 grams per liter of sodium ascorbate to each, and vortex to mix. Heat the tubes for 10 minutes at 100 degrees Celsius using marbles to prevent evaporation.Then remove the tubes from the heating block and allow them to cool to room temperature. Using a spectrometer set to 797 nanometers, obtain a calibration curve using the standards. Then, measure the absorbence of the samples against the blank, and determine the phosphate content.Begin by pipetting 800 microliters of each extruded LUV suspension into a unique two milliliter microfuse tube. Add 5.3 microliters of buffer A and 34.7 microliters of 10%mass-per-volume DDM, dissolved in buffer A, to each tube. Next, incubate the samples for three hours at room temperature with end-over-end mixing.While the samples are incubating, weigh out 400 milligrams per sample of beads in a glass beaker. Wash the beads with five milliliters per sample of methanol, stirring slowly for 10 minutes. Repeat this wash once for methanol, three times with water, and once with buffer A.During the last 30 minutes of sample incubation, dry 9.5 microliters-per-sample of NBD labeled phospholipid in a screw-cap glass tube under a stream of nitrogen.Then, add 45 microliters-per-sample of 0.1%mass-per-volume DDM in buffer A to dissolve the dried phospholipid. After incubation is complete, add the dissolved MBD labeled phospholipid solution to each sample. Then, add 0.1%DDM, buffer A, and the DDM-solubilized opsin in the desired amount, in that order, to obtain a final volume of one milliliter with a concentration of seven millimolar DDM.Reconstitution of a membrane protein is achieved by treating vesicles with sufficient detergent so that the swell will not dissolve. And that these conditions, which have to be empirically found for every lipid composition and detergent, the protein will integrate into the liposomes. Mix the samples for one hour at room temperature.Then, add 80 milligrams of the prepared polystyrene beads to each tube, and incubate again for one hour at room temperature with end-over-end mixing. Add an additional 160 milligrams of polystyrene beads to each sample, and incubate for two hours at room temperature with end-over-end mixing. When the incubation is complete, transfer the samples, leaving the beads behind, to clean, glass tubes, each containing 160 milligrams of fresh beads.Mix end-over-end overnight at four degrees Celsius, protected from light. Next, transfer the samples, without the beads, to unique microfuse tubes. Place the tubes on ice in preparation for the scramblase activity assay.Begin by adding 1950 microliters of buffer A to a plastic cubette, containing a plastic stir bar. Then, add 50 microliters of the prepared protealiposome sample. Let the sample equilibrate in the fluorescent spectrometer with constant stirring for five seconds.While the sample is equilibrating, prepare a solution of one molar dithionite in 0.5 molar unbuffered tris. Start the fluorescence monitoring with an excitation of 470 nanometers, an emission of 530 nanometers, and a slit width of 0.5 nanometers. Record for 50 seconds to achieve a stable signal.Then, add 40 microliters of one molar dithionite solution to the cubette. Record the fluorescence for at least 500 seconds. Finally, analyze the data as outlined in the text protocol.For a complete analysis of the fluorescence data, when it's to determine the fraction of vesicles that cannot be reconstituted. The recovery of proteins and lipids in the reconstituted vesicles and also the size distribution of the vesicles. In this study, opsin is reconstituted into large unilamellar vesicles to characterize its scramblase activity using a fluorescence-based assay.Vesicles are reconstituted with NBD-PC, which equally distributes between the outer and inner leaflets. Dithionite reduces the nitro group of NBD on the outside of the vesicle to a non-fluorescent amino groups resulting in a 50%reduction of fluorescence in protein-free liposomes. However, when opsin facilitates NBD movement between the inner and outer leaflet, a complete loss of fluorescence occurs.The representative results of this approach at different protein-to-phospholipid ratios can be seen here. The extend of fluorescence reduction increases with the amount of opsin reconstituted into vesicles, which ranges from zero to 4.95 micrograms. Dithionite was added at the indicated time, and NBD fluorescence was monitored for 400 seconds.Fluorescence reduction is more significant at higher protein-to-phospholipid ratios, with the largest reduction being 85%The endpoint fluorescence reduction is then used to determine the probability of scramblase activity, the likelihood of vesicles having at least one scramblase using Polson statistics. The red curve represents a mono-exponential fit for opsin. While the dashed grey lines represent mono-exponential fits for the reconstitution of opsin into vesicles as monomers, pre-formed dimers, or pre-formed tetramers.The method we describe can easily be adapted to different needs. The procedures are versatile, and can be used to identify and characterize phospholipid scramblase activity of other membrane proteins in the future. The reconstitution procedure can be applied to any membrane protein.This technique enabled us to identify opsin as the first biochemically verified phospholipid scramblase and allowed us, then, to characterize the activity of different confirmation states of the protein, and its ability to transport different lipids.

a-fluorescence-based-assay-of-phospholipid-scramblase-activity

Research

JoVE Journal

Biochemistry

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...