Name: identifying the binding proteins of small ligands with the differential radial capillary action of ligand assay (dracala)
Uploaded: 2021-03-19T14:30:00
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....

<ol>
	<li>Kalia, 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=Nucleotide,+c-di-GMP,+c-di-AMP,+cGMP,+cAMP,+(p)ppGpp+signaling+in+bacteria+and+implications+in+pathogenesis.">Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, (p)ppGpp signaling in bacteria and implications in pathogenesis.</a> Chemical Society Reviews. 42 (1), 305-341 (2013).</li><li>Camilli, A., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+small-molecule+signaling+pathways.">Bacterial small-molecule signaling pathways.</a> Science. 311 (5764), 1113-1116 (2006).</li><li>Luo, Y., 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=The+cAMP+capture+compound+mass+spectrometry+as+a+novel+tool+for+targeting+cAMP-binding+proteins:+from+protein+kinase+A+to+potassium/sodium+hyperpolarization-activated+cyclic+nucleotide-gated+channels.">The cAMP capture compound mass spectrometry as a novel tool for targeting cAMP-binding proteins: from protein kinase A to potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels.</a> Molecular &amp; Cellular Proteomics. 8 (12), 2843-2856 (2009).</li><li>Nesper, J., Reinders, A., Glatter, T., Schmidt, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+capture+compound+for+the+identification+and+analysis+of+cyclic+di-GMP+binding+proteins.">A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.</a> Journal of Proteomics. 75 (15), 4874-4878 (2012).</li><li>Laventie, B. J., 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=Capture+compound+mass+spectrometry--a+powerful+tool+to+identify+novel+c-di-GMP+effector+proteins.">Capture compound mass spectrometry--a powerful tool to identify novel c-di-GMP effector proteins.</a> Journal of Visual Experiments. (97), e51404 (2015).</li><li>Roelofs, K. G., Wang, J., Sintim, H. O., Lee, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radial+capillary+action+of+ligand+assay+for+high-throughput+detection+of+protein-metabolite+interactions.">Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (37), 15528-15533 (2011).</li><li>Zhang, Y., 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=Evolutionary+adaptation+of+the+essential+tRNA+methyltransferase+TrmD+to+the+signaling+molecule+3+',5+'-cAMP+in+bacteria.">Evolutionary adaptation of the essential tRNA methyltransferase TrmD to the signaling molecule 3 ',5 '-cAMP in bacteria.</a> Journal of Biological Chemistry. 292 (1), 313-327 (2017).</li><li>Corrigan, R. M., 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=Systematic+identification+of+conserved+bacterial+c-di-AMP+receptor+proteins.">Systematic identification of conserved bacterial c-di-AMP receptor proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 9084-9089 (2013).</li><li>Roelofs, K. G., 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=Systematic+identification+of+cyclic-di-GMP+binding+proteins+in+Vibrio+cholerae+reveals+a+novel+class+of+cyclic-di-GMP-binding+ATPases+associated+with+type+II+secretion+systems.">Systematic identification of cyclic-di-GMP binding proteins in Vibrio cholerae reveals a novel class of cyclic-di-GMP-binding ATPases associated with type II secretion systems.</a> PLoS Pathogen. 11 (10), 1005232 (2015).</li><li>Fang, X., 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=GIL,+a+new+c-di-GMP-binding+protein+domain+involved+in+regulation+of+cellulose+synthesis+in+enterobacteria.">GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.</a> Molecular Microbiology. 93 (3), 439-452 (2014).</li><li>Corrigan, R. M., Bellows, L. E., Wood, A., Grundling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp+negatively+impacts+ribosome+assembly+affecting+growth+and+antimicrobial+tolerance+in+Gram-positive+bacteria.">ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 1710-1719 (2016).</li><li>Zhang, Y., Zbornikova, E., Rejman, D., Gerdes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+(p)ppGpp+binding+and+metabolizing+proteins+of+Escherichia+coli.">Novel (p)ppGpp binding and metabolizing proteins of Escherichia coli.</a> Mbio. 9 (2), 02188 (2018).</li><li>Yang, J., 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=The+nucleotide+pGpp+acts+as+a+third+alarmone+in+Bacillus,+with+functions+distinct+from+those+of+(p)+ppGpp.">The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp.</a> Nature Communications. 11 (1), 5388 (2020).</li><li>Orr, M. W., Lee, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radial+capillary+action+of+ligand+assay+(DRaCALA)+for+high-throughput+detection+of+protein-metabolite+interactions+in+bacteria.">Differential radial capillary action of ligand assay (DRaCALA) for high-throughput detection of protein-metabolite interactions in bacteria.</a> Methods in Molecular Biology. 1535, 25-41 (2017).</li><li>Kitagawa, M., 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=Complete+set+of+ORF+clones+of+Escherichia+coli+ASKA+library+(A+complete+Set+of+E.+coli+K-12+ORF+archive):+Unique+resources+for+biological+research.">Complete set of ORF clones of Escherichia coli ASKA library (A complete Set of E. coli K-12 ORF archive): Unique resources for biological research.</a> DNA Research. 12 (5), 291-299 (2005).</li><li>Hochstadt-Ozer, J., Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+purine+utilization+in+bacteria.+V.+Inhibition+of+purine+phosphoribosyltransferase+activities+and+purine+uptake+in+isolated+membrane+vesicles+by+guanosine+tetraphosphate.">The regulation of purine utilization in bacteria. V. Inhibition of purine phosphoribosyltransferase activities and purine uptake in isolated membrane vesicles by guanosine tetraphosphate.</a> Journal of Biological Chemistry. 247 (21), 7067-7072 (1972).</li><li>Zhang, Y. E., 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=p)ppGpp+regulates+a+bacterial+nucleosidase+by+an+allosteric+two-domain+switch.">p)ppGpp regulates a bacterial nucleosidase by an allosteric two-domain switch.</a> Molecular Cell. 74 (6), 1239-1249 (2019).</li><li>Wang, B., 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=Affinity-based+capture+and+identification+of+protein+effectors+of+the+growth+regulator+ppGpp.">Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.</a> Nature Chemical Biology. 15 (2), 141-150 (2019).</li></ol>

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...

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/files/ftp_upload/62331/62331fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32P-&#945;-GTP</td>
			<td>Perkinelmer</td>
			<td>BLU006X250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>96 x pin tool</td>
			<td>V&#38;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&#8211;299. https://doi.org/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>&#8805;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., &#8805; 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 &#946;-d-1-thiogalactopyranoside (IPTG)</td>
			<td>SIGMA</td>
			<td>I6758</td>
			<td>Isopropyl &#946;-D-thiogalactoside</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>Invitrogen - Thermo Fisher</td>
			<td>12795027</td>
			<td>Miller&#39;s LB Broth Base</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>SIGMA</td>
			<td>L4949</td>
			<td>from chicken egg white; BioUltra, lyophilized powder, &#8805;98%</td>
		</tr>
		<tr>
			<td>MgCl2 (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 &#956;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>&#946;-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 targe...

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 ...

Small signaling molecules target various effector proteins to control bacterial virulence and human biology. However, these effector proteins are challenging to identify. As a systems biology tool, DRaCALA allows a feasible, rapid, and highly sensitive identification of the unknown effector proteins by using an OVium library.DRaCALA could be used to study any small signaling molecule as long as it can be labeled with either radioactive isotope or fluorescent dyes. Start by inoculating E.coli into 1.5 milliliters of LB supplemented with 25 micrograms per milliliter chloramphenicol in each well of 96-well deep well plates for an overnight incubation at 30 degrees Celsius and 160 rotations per minute. The next morning, treat the overnight cultures with 500 micromolar IPTG for six hours at 30 degrees Celsius to induce protein expression.At the end of the incubation, pellet the cells by centrifugation, remove the supernatant, and freeze the samples at minus 80 degrees Celsius. To initiate lysis, resuspend the pellets in 150 microliters of lysis buffer one per well for a 30-minute incubation at minus 80 degrees Celsius before thawing the cells for 20 minutes at 37 degrees Celsius. The lysate should then be stored at minus 80 degrees Celsius before use.To complete the lysis of cells with overexpressed proteins, resuspend the samples in 40 milliliters of ice cold lysis buffer two and sonicate the samples at a 60%amplitude at two seconds on four seconds off for eight minutes, then clear the lysates by centrifugation. While the samples are being centrifuged, add 500 microliters of homogenized nickel-NTA resin to a standing polypropylene chromatography column. After 15 minutes, wash the settled resin two times with 15 milliliters of ultrapure water and one time with 15 milliliters of lysis buffer two.At the end of the centrifugation, load the cleared lysate supernatant onto the column. When all the lysate has flowed through the column, wash the column with 30 milliliters of washing buffer. To elute the proteins, add 400 microliter volume of elution buffer to the column and repeat elution three times.Another 300 microliter volume of the elution buffer to repeat elution three times and combine the eluted proteins in a final volume of 700 microliters. For gel filtration of the eluted sample, after washing a size exclusion column with 25 milliliters of freshly prepared gel filtration buffer, load the entire volume of eluted protein onto the column by using a 500 microliter loop. Run at 500 microliters per minute and collect two to three 500 microliter volume fractions of the respective proteins.Then use individual spin columns to concentrate each protein and use the Bradford assay kit to measure the protein concentrations according to standard protocols. To synthesize the phosphorus 32 labeled guanosine pentaphosphate, assemble a small-scale Relseq reaction in a screw capped tube as outlined in the table and incubate the reaction in a ThermoMixer for one hour at 37 degrees Celsius and five minutes at 95 degrees Celsius, followed by five minutes on ice. At the end of the incubations, spin down the precipitated protein by centrifugation and transfer the synthesized phosphorus 32 labeled guanosine pentaphosphate containing supernatant to a new screw capped tube.For phosphorus 32 guanosine tetraphosphate synthesis, add one micromolar GppA to half of the phosphorus 32 labeled guanosine pentaphosphate product in a new screw capped tube and incubate the reaction for 10 minutes at 37 degrees Celsius, five minutes at 95 degrees Celsius, and five minutes on ice. At the end of the incubation, spin down the precipitate by centrifugation and transfer the phosphorus 32 labeled guanosine tetraphosphate containing supernatant to a new tube. To analyze the isolated target proteins, run one microliter of each sample on a thin layer chromatography plate using 1.5 molar monopotassium phosphate as the mobile phase.After the analysis, place the dried plate in a transparent plastic folder and expose the plate to a storage phosphor screen for five minutes before visualizing and quantifying the data on a phosphor imager. For DRaCALA screening of the target proteins, add 20 microliters of the thawed wholesale lysates to individual wells of a 95-well V-bottom microtiter plate and add 2.5 units of Serratia marcescens endonuclease to each well. After 15 minutes at 37 degrees Celsius, place the lysates on ice for 20 minutes.Next, mix equal volumes of phosphorus 32 labeled guanosine pentaphosphate and guanosine tetraphosphate and add 1X lysis buffer one to the mixture to obtain a four nanomolar guanosine pentaphosphate solution. Using a multi-channel pipette and filtered pipette tips, mix 10 microliters of the guanosine pentaphosphate mixture with the cell lysate for a five-minute incubation at room temperature. At the end of the incubation, wash a 96X pin tool three times in a 0.01%solution of non-ionic detergent for 30 seconds, followed by 30 seconds of drying on a paper towel per wash before placing the pin tool in the 96-well sample plate.After 30 seconds, lift the pin tool straight up and place it straight down on a nitrocellulose membrane for 30 seconds. After five minutes of drying, place the nitrocellulose membrane in a transparent plastic folder for storage phosphor screen exposure and visualization by phosphor imaging as demonstrated. To quantify and identify potential target proteins, in the analysis software associated with the phosphor imager, open the gel file of the visualized plates.To define the spots to be analyzed, use the array analysis function to set up a 12 column by eight row grid. To circumscribe the outer edge of the hole spots, define big circles, export the volume plus background and area of the defined big circles to a spreadsheet. To circumscribe the small inner dots, size down the defined circles, export the volume plus background and area of the defined small circles and save all the data in the spreadsheet.Position circles to overlap with spots as necessary, resizing the slightly bigger than actual spots. Use the equation to calculate the binding fractions in the spreadsheet and plot the data. Then identify the potential binding proteins in the wells that show high binding fractions compared to the majority of other wells.In this representative analysis, a plate with relatively low background binding signals in most wells can be observed. The positive binding signal in well H3 exhibited a binding fraction that was much higher than that observed for the other wells due to overexpression of the guanosine pentaphosphate binding protein Hpt. In the representative plates, several wells showed a relatively higher background binding signals as indicated by the relatively strong inner dots observed in many wells, as well as the consistently high binding fractions observed after quantification.Notably, some targets also gave variable binding fractions, such as the false positives. To clarify further, researchers used purified proteins to test the binding again. Once the target proteins are identified, one can purify them to homogeneity and confirm interaction strength and specificity by using either DRaCALA, isothermal titration calorimetry, or other methods.Identifying the target proteins of small signaling molecules paves the way to an in-depth understanding of the roles emerging from bacterial virulence to human biology.

Research

JoVE Journal

Biochemistry

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 ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

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 ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...