Name: identifying the binding proteins of small ligands with the differential radial capillary action of ligand assay (dracala)
Uploaded: 2021-03-19T14:30:00.000+00:00
Duration: 9 min 26 s
Description: Watch this Scientific Journal Video about Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA at JoVE.com

The work is supported by an NNF Project Grant (NNF19OC0058331) to YEZ, and the European Union&#8217;s Horizon 2020 research and innovation programme under the Marie Sk&#322;odowska-Curie grant agreement (N&#186; 801199) to MLS.
<img alt="Equation 1" src="/files/ftp_upload/62331/62331img1.jpg" />

1. Preparation of whole cell lysates
<ol>
	<li>Inoculate the E. coli K-12 ASKA ORFeome collection strains15 into 1.5 mL Lysogeny broth (LB) containing 25 &#181;g/mL chloramphenicol in 96-well deep well plates. Grow overnight (O/N) for 18 h at 30 &#176;C with shaking at 160 rpm. On the next day, add isopropyl &#946;-d-1-thiogalactopyranoside (IPTG) (final 0.5 mM) to the O/N cultures to induce protein expression at 30 &#176;C for 6 h.</li>
	<li>Pellet cells at 500 x g for 10 min. Freeze the pellets at -80 &#176;C until use. To lyse the cells, add 150 &#181;L of lysis buffer L1 (40 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2, supplemented with 2 mM phenylmethylsulfonyl fluoride (PMSF), 40 &#181;g/mL DNase 1, and 0.5 mg/mL lysozyme) to resuspend the pellet.</li>
	<li>Freeze the cells at -80 &#176;C for 30 min, and then thaw at 37 &#176;C for 20 min. Repeat this cycle three times to lyse the cells. Store the lysates at -80 &#176;C before use.</li>
</ol>
2. Purification of Relseq and GppA
NOTE: The recombinant proteins Relseq from Streptococcus equisimilis and GppA from E. coli K-12 are used to synthesize the radiolabeled pppGpp and ppGpp, respectively.
<ol>
	<li>Grow and collect cells overexpressing each protein.
	<ol>
		<li>Grow the E. coli BL21 DE3 strain up to exponential phase (optical density (OD) ~0.3-0.4) in LB broth, and spin down 1 mL of culture at 6000 x g for 5 min. Decant the supernatant, and resuspend the cells with 100 &#181;L of ice-cold TSB broth (LB broth supplemented with 0.1 g/mL PEG3350, 0.05 mL/mL dimethylsulfoxide, 20 mM MgCl2).</li>
		<li>Mix the plasmids bearing the histidine-tagged relseq and gppA, each 100 ng, with the above cell suspensions in TSB, and incubate on ice for 30 min. Heat-shock the cells at 42 &#176;C for 40 s. Place the mixture on ice for 2 min, and add 1 mL of LB broth at room temperature to allow the cells to recover for 1 h at 37 &#176;C with agitation at 160 rpm.</li>
		<li>Plate the recovered cells on LB agar plates supplemented with the corresponding antibiotics (Relseq:100 &#181;g/mL ampicillin; GppA: 30 &#181;g/mL kanamycin).On the next day, inoculate the colonies in LB broth to start O/N precultures of both strains at 37 &#176;C.</li>
		<li>After 18 h, inoculate 500 mL of LB medium with 10 mL of the O/N cultures and the corresponding antibiotics. Grow the cultures by shaking at 160 rpm at 37 &#176;C. When the OD600nm reaches 0.5-0.7, induce protein expression by adding 0.5 mM IPTG and growing for 3 h at 30 &#176;C with shaking at 160 rpm.</li>
		<li>Collect the cells by spinning at 6084 x g for 10 min at 4 &#176;C. Resuspend the pellet in 20 mL of ice-cold 1x phosphate-buffered saline (PBS), and re-centrifuge at 1912 x g for 20 min at 4 &#176;C. Decant the supernatant, and freeze the pellets at -20 &#176;C before use.</li>
	</ol>
	</li>
	<li>Nickel-nitrilotriacetic acid (Ni-NTA) affinity purification 
	&#8203;NOTE: From this point onwards, ensure that the samples are cold.
	<ol>
		<li>Add 40 mL of ice-cold lysis buffer L2 (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM imidazole, 10 mM &#946;-mercaptoethanol supplemented with protease inhibitors (EDTA-free tablet; see the Table of Materials) to resuspend the pellet. Lyse the cells via sonication (60% amplitude, 2 s ON/ 4 s OFF for 8 min ON). Clear the lysate by spinning at 23,426 x g for 40 min at 4 &#176;C, and continue with the supernatant for the purification.</li>
		<li>During the above centrifugation, prepare the Ni-NTA resin.
		<ol>
			<li>Transfer 500 &#181;L of homogenized Ni-NTA resin into a standing polypropylene chromatography column, and let it settle for 15 min and the storage solution drain through. Wash the resin with 15 mL of ultrapure water twice, and then wash the column with 15 mL of the lysis buffer L2.</li>
		</ol>
		</li>
		<li>Load the cleared supernatant of cell lysate from step 1 onto the column, and let it flow through. Wash the column with 30 mL of washing buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 20 mM imidazole).</li>
		<li>Elute the proteins with 400 &#181;L of the elution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 500 mM imidazole) three times. Then, repeat elution with another 300 &#181;L of the elution buffer. Combine the eluted proteins to a final volume of 700 &#181;L.</li>
	</ol>
	</li>
	<li>Gel filtration
	<ol>
		<li>Prepare gel filtration buffer (50 mM Tris, pH 7.5; 200 mM NaCl; 5% glycerol). Wash the size exclusion column with one column volume (25 mL) of the gel filtration buffer.</li>
		<li>Load the above 700 &#181;L sample by using a 500 &#181;L loop, run at 0.5 mL/min, and collect 2-3 fractions, each of 0.5 mL volume, containing the respective proteins.</li>
		<li>Combine and concentrate the fractions containing each of the proteins using a spin column, and measure the protein concentration using the Bradford assay.</li>
	</ol>
	</li>
</ol>
3. Synthesis of 32P-labeled pppGpp and ppGpp
<ol>
	<li>Assemble a small-scale Relseq reaction in a screw cap tube (see Table 1). 
	NOTE: Work with radioactive reagents only in a licensed place and with personal protective equipment.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Volume (&#956;L)</td>
		</tr>
		<tr>
			<td></td>
			<td>Small scale</td>
			<td>Large scale</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Relseq buffer*</td>
			<td>2</td>
			<td>50</td>
		</tr>
		<tr>
			<td>ATP (8 mM final)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Relseq (4 &#956;M final)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>32P-&#945;-Guanosine triphosphate (GTP) (final 120 nM) (CAUTION)</td>
			<td>0.2</td>
			<td>5</td>
		</tr>
		<tr>
			<td>total&#160;</td>
			<td>20</td>
			<td>500</td>
		</tr>
	</tbody>
</table>
Table 1: Assembling information for the small- and large-scale synthesis reactions of 32P-labeled pppGpp. *10x Relseq buffer contains 250 mM Tris-HCl, pH 8.6; 1M NaCl; 80 mM MgCl2. Abbreviation: pppGpp = guanosine pentaphosphate.
<ol start="2">
	<li>Incubate the tube at 37 &#176;C in a thermomixer for 1 h, then at 95 &#176;C for 5 min, and place on ice for 5 min. Spin down the precipitated protein at 15,700 x g for 5 min, and transfer the supernatant (synthesized 32P-pppGpp) to a new screw cap tube.</li>
	<li>To synthesize 32P-ppGpp from 32P-pppGpp, transfer half of the 32p-pppGpp product to a new screw cap tube, and add 1 &#181;M GppA. Incubate the tube at 37 &#176;C for 10 min, at 95 &#176;C for 5 min, and then place on ice for 5 min.</li>
	<li>Spin down the precipitated protein at 15,700 x g for 5 min, and transfer the supernatant (synthesized 32P-ppGpp) to a new screw cap tube.</li>
	<li>Analyze the 32P-pppGpp and 32P-ppGpp by running 1 &#181;L of the samples on a thin layer chromatography (TLC) plate (polyethyleneimine-modified cellulose TLC plates) using the 1.5 M KH2PO4, pH 3.4, as mobile phase. 
	NOTE: Use the &#945;-32P-labeled guanosine 5&#39;-triphosphate (32P-&#945;-GTP) as control.</li>
	<li>Dry the TLC plate completely, place it between a transparent plastic folder, and expose it to a storage phosphor screen for 5 min. Visualize and quantify the signals by using a phosphorimager. 
	&#8203;NOTE: When the ratios of 32P-pppGpp and 32P-ppGpp are higher than 85%, a large-scale reaction (500 &#181;L, sufficient for screening 20 96-well plates) could be assembled and synthesized by using Table 1.</li>
</ol>
4. DRaCALA screening of the target proteins of (p)ppGpp
<ol>
	<li>Thaw and transfer 20 &#181;L of whole cell lysates to a 96-well V-bottom microtiter plate. Add 2.5 U/well of endonuclease from Serratia marcescens, and incubate at 37 &#176;C for 15 min to reduce lysate viscosity. Place the lysates on ice for 20 min.</li>
	<li>Mix the 32P-pppGpp and 32P-ppGpp in a 1:1 ratio, and add 1x lysis buffer L1 to make the final concentration of (p)ppGpp equal to 4 nM. 
	NOTE: Given the chemical similarity between pppGpp and ppGpp, a mix of both chemicals will simplify the screening process.</li>
	<li>Use a multichannel pipette and filtered pipette tips to add and mix 10 &#181;L of the (p)ppGpp mixture with the cell lysate. Incubate at room temperature (RT) for 5 min.</li>
	<li>Wash the 96 x pin tool by placing in 0.01% solution of non-ionic detergent for 30 s, and dry on a tissue paper for 30 s. Repeat the washing of the pin tool 3x.</li>
	<li>Place the pin tool in the above 96-well sample plates, and wait for 30 s. Lift the pin tool straight up, and place it straight down on a nitrocellulose membrane for 30 s. 
	&#8203;NOTE: If a spot is missing, spot 2 &#181;L of the corresponding samples with a pipette and filtered tips. It is advisable to make a duplicate spot of the same sample as indicated below.</li>
	<li>Dry the membrane for 5 min at RT. Place the membrane between a transparent plastic folder, and expose it to a storage phosphor screen for 5 min. Visualize by using a phosphorimager.</li>
</ol>
5. Quantification and identification of potential target proteins
<ol>
	<li>Use the analysis software associated with the phosphorimager to open the .gel file of the visualized plates. Use the Array analysis function to define the 96 spots by setting up a grid of 12 columns x 8 rows.</li>
	<li>Define big circles to circumscribe the outer edge of the whole spots (see Figure 1B). Export the Volumn+Background and Area of the defined big circles, and save in a spreadsheet. 
	NOTE: If required, reposition each individual circle to perfectly overlap with the spots, and resize each individual circle to make it slightly bigger than the actual spot.</li>
	<li>Size down the defined circles to circumscribe the small inner dots. Export the Volumn+Background, and Area of the defined small circles, and save in a spreadsheet.</li>
	<li>Calculate the binding fractions in the spreadsheet by using the equation in Figure 1B, and plot the data. Identify the potential binding proteins in the wells that show high binding fractions in comparison to the majority of other wells.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62331/62331fig02.jpg" /> 
Figure 2:&#160;Overall workflow of the DRaCALA screening process. Protein production from an Escherichia coli ASKA collection is induced, and the cells are lysed. Meanwhile, the recombinant proteins Relseq-His and GppA-His are purified and used to synthesize 32P-labeled pppGpp and ppGpp from 32P-&#945;-GTP. The radioactively labeled (p)ppGpp molecules are then mixed with the lysates, and a 96 pin-tool is used to spot the mixtures onto a nitrocellulose membrane for subsequent exposure to a phosphor storage screen, imaging, and quantification of the radioactive signals. Abbreviations: DRaCALA = Differential Radial Capillary Action of Ligand Assay; (p)ppGpp = guanosine penta- and tetraphosphates; RT = room temperature; IPTG = isopropyl &#946;-d-1-thiogalactopyranoside; GTP = guanosine 5&#39;-triphosphate; SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis ; TLC = thin layer chromatography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62331/62331fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have no conflict of interest to disclose.

One of the critical steps in performing DRaCALA screening is to obtain good whole cell lysates. First, the tested proteins should be produced in large amounts and in soluble forms. Second, the lysis of cells should be complete, and the viscosity of the lysate must be minimal. The inclusion of lysozyme and the use of three cycles of freeze-thaw are often enough to lyse cells completely. However, the released chromosomal DNA makes the lysate viscous and generates high background binding signal, resulting in false positives as shown in Figure 3B,C. To mitigate this, DNase 1 and/or endonuclease from S. marcescens can be used, and the samples can be incubated for a longer time to degrade the DNA.
Both negative (empty plasmid vector) and positive (known target protein) controls should be used to optimize the conditions for whole cell lysate preparation and the DRaCALA binding assay before performing a large-scale screening. When such an optimal condition is found, it becomes unnecessary to include the controls in the actual screening of each plate. This is because the screening procedure is very short and because the majority of the proteins in a plate are expected to not bind to (p)ppGpp. Therefore, these proteins serve as negative controls when quantifying the binding fractions (Figure 3).
The production of purer 32P-labeled pppGpp and ppGpp is also essential for DRaCALA screening. For instance, the conversion ratio from GTP to pppGpp could vary. Thus, it is important to optimize the pH, the concentrations of magnesium and enzymes used, and the reaction time. Small-scale reactions are therefore important to identify the best condition before setting up a large-scale reaction.
DRaCALA clearly has some advantages and disadvantages (see Roelofs et al.9 for more discussion) when compared to other techniques such as the capture compound technique. First, DRaCALA uses the 32P-labeled (p)ppGpp, which does not affect the chemical structure of (p)ppGpp, thereby preserving its interaction with potential target proteins. Second, once the 32p-(p)ppGpp and cell lysates are prepared, it takes only a week to screen ~60 plates of the E. coli ORFeome library using DRaCALA. Indeed, the short experimental procedure (section 4) allowed the identification of even the proteins that degrade the (p)ppGpp (MutT, NudG)12,17, which the capture compound technique missed18.
However, the use of DRaCALA requires an ORFeome expression library, which is only available for a limited number of model organisms. Besides, the affinity purification tags, which are designed to facilitate protein purification in the ORFeome library, sometimes affect the expression or proper folding of the proteins, producing some false negatives. A new ORFeome library will ideally require the determination as to whether the majority (&#62;80%) of the proteins are expressed in soluble form and in adequate amounts. Such a test also allows the researchers to evaluate the coverage and effectiveness of the DRaCALA screening results.
Despite these limitations, DRaCALA is a powerful technique to successfully uncover novel target proteins of several nucleotide secondary messengers. In principle, DRaCALA could be used for studying any small ligand if it could be labeled by using either radioactive isotopes or even fluorescent dyes.

Bacteria use several small signaling molecules to adapt to constantly changing environments1,2. For example, the autoinducers, N-acylhomoserine lactones and their modified oligopeptides, mediate the intercellular communication among bacteria to coordinate population behavior, a phenomenon known as quorum sensing2. Another group of small signaling molecules is the NSMs, including the widely studied cyclic adenosine monophosphate (cAMP), cyclic di-AMP, cyclic di-guanosine monophosphate (cyclic di-GMP), and guanosine penta- and tetra phosphates (p)ppGpp1. Bacteria produce these NSMs as a response to a variety of different stress conditions. Once produced, these molecules bind to their target proteins and regulate several different physiological and metabolic pathways to cope with the encountered stresses and enhance bacterial survival. Therefore, identification of the target proteins is an unavoidable prerequisite for deciphering the molecular functions of these small molecules.
The past decade has witnessed a boom of knowledge of these small signaling molecules, mainly due to several technical innovations that unveiled the target proteins of these small molecules. These include the capture compound technique3,4,5, and the differential radial capillary action of ligand assay (DRaCALA)6 to be discussed in this paper.
Invented by Vincent Lee and co-workers in 20116, DRaCALA deploys the ability of a nitrocellulose membrane to differentially sequester free and protein-bound ligands. Molecules such as proteins cannot diffuse on a nitrocellulose membrane, while small ligands, such as the NSMs, are able to. By mixing the NSM (e.g., ppGpp) with the protein to be tested and spotting them on the membrane, two scenarios can be expected (Figure 1): If (p)ppGpp binds to the protein, the radiolabeled (p)ppGpp will be retained in the center of the spot by the protein and will not diffuse outward, giving an intense small dot (i.e., strong radioactive signal) under a phosphorimager. However, if (p)ppGpp does not bind to the protein, it will diffuse freely outward to produce a large spot with uniform background radioactive signal.
Furthermore, DRaCALA can detect the interaction between a small molecule and an unpurified protein in a whole cell lysate if the protein is present in a sufficient amount. This simplicity allows the use of DRaCALA in rapidly identifying protein targets by using an ORFeome expression library. Indeed, target proteins of cAMP7, cyclic di-AMP8, cyclic di-GMP9,10, and (p)ppGpp11,12,13 have been systematically identified by using DRaCALA. This video article uses (p)ppGpp as an example to demonstrate and describe the critical steps and considerations in performing a successful DRaCALA screening. Of note, a more thorough description of DRaCALA14 is highly recommended to read in combination with this article before performing DRaCALA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62331/62331fig01.jpg" /> 
Figure 1:&#160;The principle of DRaCALA. (A) Schematic of the DRaCALA assay. See the text for details. (B) Quantification and calculation of the binding fraction. See the text for details. Briefly, the DRaCALA spots will be analyzed by drawing two circles that circumscribe the whole spot and the inner dark dot (i.e., the retained (p)ppGpp due to the binding of the tested protein). The specific binding signal is the radioactive signal of the inner circle (S1) after subtracting the non-specific background signal (calculated by A1 &#215; ((S2-S1)/(A2-A1))). The binding fraction is the specific binding signal divided by the total radioactive signal (S2). Abbreviations: DRaCALA = Differential Radial Capillary Action of Ligand Assay; (p)ppGpp = guanosine penta- and tetraphosphates; RT = room temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62331/62331fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;sup&gt;32&lt;/sup&gt;P-&amp;alpha;-GTP</td><td>Perkinelmer</td><td>BLU006X250UC</td><td></td></tr><tr><td>96 x pin tool</td><td>V&amp;amp;P Scientific</td><td>VP 404</td><td>96 Bolt Replicator, on 9 mm centers, 4.2 mm Bolt Diameter, 24 mm long</td></tr><tr><td>96-well V-bottom microtiter plate</td><td>Sterilin</td><td>MIC9004</td><td>Sterilin Microplate V Well 611V96</td></tr><tr><td>Agar</td><td>OXOID - Thermo Fisher</td><td>LP0011</td><td>Agar no. 1</td></tr><tr><td>ASKA collection strain</td><td>NBRP, SHIGEN, JAPAN</td><td></td><td>Ref: DNA Research, Volume 12, Issue 5, 2005, Pages 291&amp;ndash;299. https://doi-org.remotexs.ntu.edu.sg/10.1093/dnares/dsi012</td></tr><tr><td>Benzonase</td><td>SIGMA</td><td>E1014-25KU</td><td>genetically engineered endonuclease from Serratia marcescens</td></tr><tr><td>Bradford Protein Assay Dye</td><td>Bio-Rad</td><td>5000006</td><td>Reagent Concentrate</td></tr><tr><td>DMSO</td><td>SIGMA</td><td>D8418</td><td>&amp;ge;99.9%</td></tr><tr><td>DNase 1</td><td>SIGMA</td><td>DN25-1G</td><td></td></tr><tr><td>gel filtration10x300 column</td><td>GE Healthcare</td><td>28990944</td><td>contains 20% ethanol as preservative</td></tr><tr><td>Glycerol</td><td>PanReac AppliChem</td><td>122329.1214</td><td>Glycerol 87% for analysis</td></tr><tr><td>Hypercassette</td><td>Amersham</td><td>RPN 11647</td><td>20 x 40 cm</td></tr><tr><td>Imidazole</td><td>SIGMA</td><td>56750</td><td>puriss. p.a., &amp;ge; 99.5% (GC)</td></tr><tr><td>IP Storage Phosphor Screen</td><td>FUJIFILM</td><td>28956474</td><td>BAS-MS 2040 20x 40 cm</td></tr><tr><td>Isopropyl &amp;beta;-d-1-thiogalactopyranoside (IPTG)</td><td>SIGMA</td><td>I6758</td><td>Isopropyl &amp;beta;-D-thiogalactoside</td></tr><tr><td>Lysogeny Broth (LB)</td><td>Invitrogen - Thermo Fisher</td><td>12795027</td><td>Miller&#039;s LB Broth Base</td></tr><tr><td>Lysozyme</td><td>SIGMA</td><td>L4949</td><td>from chicken egg white; BioUltra, lyophilized powder, &amp;ge;98%</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (Magnesium chloride)</td><td>SIGMA</td><td>208337</td><td></td></tr><tr><td>MilliQ water</td><td></td><td></td><td>ultrapure water</td></tr><tr><td>multichannel pipette</td><td>Thermo Scientific</td><td>4661110</td><td>F1 - Clip Tip; 1-10 ul, 8 x channels</td></tr><tr><td>NaCl</td><td>VWR Chemicals</td><td>27810</td><td>AnalaR NORMAPUR, ACS, Reag. Ph. Eur.</td></tr><tr><td>Ni-NTA Agarose</td><td>Qiagen</td><td>30230</td><td></td></tr><tr><td>Nitrocellulose Blotting Membrane</td><td>Amersham Protran</td><td>10600003</td><td>Premium 0.45 um 300 mm x 4 m</td></tr><tr><td>PBS</td><td>OXOID - Thermo Fisher</td><td>BR0014G</td><td>Phosphate buffered saline (Dulbecco A), Tablets</td></tr><tr><td>PEG3350 (Polyethylene glycol 3350)</td><td>SIGMA</td><td>202444</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>SIGMA</td><td>93482</td><td>Phenylmethanesulfonyl fluoride solution - 0.1 M in ethanol (T)</td></tr><tr><td>Phosphor-imager</td><td>GE Healthcare</td><td>28955809</td><td>Typhoon FLA-7000 Phosphor-imager</td></tr><tr><td>Pipette Tips, filtered</td><td>Thermo Scientific</td><td>94410040</td><td>ClipTip 12.5 &amp;mu;l nonsterile</td></tr><tr><td>Poly-Prep Chromatography column</td><td>Bio-Rad</td><td>7311550</td><td>polypropylene chromatography column</td></tr><tr><td>Protease inhibitor Mini</td><td>Pierce</td><td>A32955</td><td>Tablets, EDTA-free</td></tr><tr><td>screw cap tube</td><td>Thermo Scientific</td><td>3488</td><td>Microcentifuge Tubes, 2.0 ml with screw cap, nonsterile</td></tr><tr><td>SLS 96-deep Well plates</td><td>Greiner</td><td>780285</td><td>MASTERBLOCK, 2 ML, PP, V-Bottom, Natural</td></tr><tr><td>spin column</td><td>Millipore</td><td>UFC500396</td><td>Amicon Ultra -0.5 ml Centrifugal Filters</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>5382000015</td><td>Thermomixer C</td></tr><tr><td>TLC plate (PEI-cellulose F TLC plates)</td><td>Merck Millipore</td><td>105579</td><td>DC PEI-cellulose F (20 x 20 cm)</td></tr><tr><td>Tris</td><td>SIGMA</td><td>BP152</td><td>Tris Base for Molecular Biology</td></tr><tr><td>Tween 20</td><td>SIGMA</td><td>P1379</td><td>viscous non-ionic detergent</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>SIGMA</td><td>M3148</td><td>99% (GC/titration)</td></tr></tbody></table>

identifying the binding proteins of small ligands with the differential radial capillary action of ligand assay (dracala)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Following the above-described protocol will typically yield two types of results (Figure 3).
Figure 3A shows a plate with relatively low background binding signals (binding fractions &#60; 0.025) from the majority of wells. The positive binding signal from the well H3 gives a binding fraction of ~0.35 that is much higher than that observed for the other wells. Even without quantification, well H3 is remarkable, suggesting that a target protein expressed in well H3 binds to either pppGpp, ppGpp, or both. Indeed, the protein overexpressed in well H3 is the hypoxanthine phosphoribosyltransferase Hpt, which is known to bind (p)ppGpp12,16.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62331/62331fig03.jpg" />
Figure 3:&#160;Representative DRaCALA screening plates (to the left) and quantification (to the right). (A) DRaCALA spots of the ASKA Plate-50. The only positive hit, Hpt, gave a strong binding signal standing out in both the spot and the quantitation diagram. (B,C) Two replicate DRaCALA spots of the rearranged plate 31. Black broken circles and arrows indicate the true target proteins of (p)ppGpp, while the red broken circles indicate the false positives. See the text for details. Abbreviations: DRaCALA = Differential Radial Capillary Action of Ligand Assay; (p)ppGpp = guanosine penta- and tetraphosphates; RT = room temperature; IPTG = isopropyl &#946;-d-1-thiogalactopyranoside; GTP = guanosine 5&#39;-triphosphate; SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis ; TLC = thin layer chromatography; Hpt = hypoxanthine phosphoribosyltransferase; PrfC = peptide chain release factor RF3; NadR = NMN adenylyltransferase; HflX = translational GTPase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62331/62331fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The other typical result of the screening is shown in&#160;Figure 3B,C. In this plate, several wells showed relatively higher background binding signals than those in&#160;Figure 3A.&#160;This is clearly visible from the relatively strong inner dots for many wells. Quantification also showed that many wells have binding fractions in the range of 0.02-0.04. A higher background of the binding signal is likely caused by the whole cell lysate being viscous despite the treatments with both DNase I and the endonuclease from S. marcescens, which degrade the released chromosomal DNA. For plates such as this one, it is important to compare the two replicate spots of the plate (step 4.5; Figure 3B,C). Quantification of both plates shows that the authentic positive targets (black circles, wells A10 PrfC, B11 NadR) tend to give consistently high binding fractions.
Notably, some true targets could also give variable binding fractions (Well D5, HflX12) such as the false positives (red circles). The reason for this variability lies in the fact that not all proteins in a library are expressed in soluble form and in required amounts. If the concentration of a protein is close to or just below the Kd value, variable binding results can be expected, even for the true targets. Indeed, large amounts of soluble HflX protein were not obtained from the ASKA strain12. To determine whether these proteins are true binders or not, the potential proteins must be purified to homogeneity and the binding confirmed by using a higher concentration (50-100 &#181;M) of the proteins.
Via this screening, 9 out of 20 known target proteins of (p)ppGpp12 were identified (see the discussion section), validating the usefulness of DRaCALA for this task. Additionally, 12 new targets of (p)ppGpp were discovered and confirmed, demonstrating that DRaCALA is a powerful technique to uncover novel target proteins of (p)ppGpp.

Watch this Scientific Journal Video about Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA at JoVE.com

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

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3.4K Views.  University of Copenhagen. The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

Video: Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

Chemistry

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ failure, which is associated with high morbidity and mortality. Collagen is a key driver of fibrosis, with type I and type III collagen being the primary types involved in many fibrotic diseases. Unlike conventional protocols used to immobilize other proteins (e.g., elastin, albumin, fibronectin, etc.), comprehensive protocols to reproducibly immobilize different types of collagens in order to produce stable coatings are not readily available. Immobilizing collagen is surprisingly challenging because multiple experimental conditions may affect the efficiency of immobilization, including the type of collagen, the pH, the temperature, and the type of microplate used. Here, a detailed protocol to reproducibly immobilize and quantify type I and III collagens resulting in&#160;stable and reproducible gels/films is provided. Furthermore, this work demonstrates how to perform, analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, including&#160;the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

This work presents an optimized protocol to reproducibly immobilize and quantify type I and III collagen onto microplates, followed by an improved in vitro binding assay protocol to study collagen-compound interactions using a time-resolved fluorescence method. The subsequent step-by-step data analysis and data interpretation are provided.

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Biology

Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins

Source: Schicketanz, M. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62331">Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA).</a> J. Vis. Exp. (2021)
This video demonstrates the differential radial capillary action of ligand assay &#8212; a technique for systematic high-throughput screening of intracellular nucleotide second messenger-binding proteins. This technique allows for direct binding of the messengers to the overexpressed target protein in the cell lysate &#8212; bypassing the need for protein purification.

Source: Schicketanz, M. L., et al. Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay ...

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Biochemical assays

Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions

Source: Schoofs, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57271">A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4.</a> J. Vis. Exp. (2018).
In this video, we describe a flow cytometry-based competitive binding assay to detect the interactions between the CXC Chemokine Receptor 4 (CXCR4) and its fluorescently-labeled natural ligand CXC Chemokine Ligand 12 (CXCL12), in the presence of a CXCR4-targeting small molecule. Incubating CXCR4-expressing cells with lower small molecule concentrations allows CXCR4-CXCL12 binding to some extent, which progressively declines upon a gradual increase in small molecule concentrations.

Source: Schoofs, G., et al. A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to ...