We thank Fabrice Agou and Alix Boucharlat from the Chemogenomic and Biological Screening Platform, Center for Technological Resources and Research (C2RT) at Institut Pasteur Paris for helpful suggestions on the protocol, all members of the BGPB Unit for support and scientific discussions, and Simon Legood for critical reading of the manuscript. Work was funded by Global Care Initiatives of the Institute Carnot Infectious diseases and the Institute Carnot Microbes and Health (15 CARN 0017-01 and 16 CARN 0023-01).

1. Enzyme and substrate preparation
<ol>
	<li>Purification of enzyme
	<ol>
		<li>Produce and purify Lnt enzyme from detergent solubilized membranes as described previously4,8. Briefly, induce expression of the lnt-strep gene, encoding Lnt with a C-terminal Strep tag, at OD600 of 0.6 with anhydrous tetracycline (200 ng/mL) at 37 &#176;C for 16 h.</li>
		<li>Harvest cells by centrifugation at 4,000 x g for 10 min and discard the supernatant.</li>
		<li>Resuspend the cell pellet in buffer WA (20 mM Tris-HCl pH 8, 150 mM NaCl, 0.5 mM EDTA) to 100 OD600 units per mL.</li>
		<li>Break the cells by two passages through a French pressure cell press at 10,000 psi.</li>
		<li>Remove unbroken cells and debris by centrifugation at 13,000 x g for 20 min. Keep the supernatant and discard the pellet.</li>
		<li>Centrifuge the supernatant at 120,000 x g for 60 min and collect the membrane vesicles (translucent brown colored pellet). Discard the supernatant.</li>
		<li>Solubilize the integral membrane proteins from the membrane pellet with 1% (w/v) n-Dodecyl &#946;-D-maltoside (DDM) in buffer WA for 30 min at room temperature (RT). Centrifuge and remove insolubilized material at 120,000 x g for 30 min. Use the supernatant fraction for purification.</li>
		<li>Purify Lnt-strep on an affinity chromatography column (see Table of Materials) and an S400 gel filtration column (see Table of Materials) as described by Nozeret et al.8.</li>
		<li>Store the purified enzyme in buffer WA containing 0.05% DDM and 10% glycerol at -80 &#176;C. 
		NOTE: The enzyme is stable for over 5 years.</li>
	</ol>
	</li>
	<li>Preparation of alkyne-phospholipid and biotinylated Fibroblast Stimulating Ligand (FSL-1-biotin) substrates
	<ol>
		<li>Aliquot 100 &#181;L of custom-synthesized alkyne-POPE (1-hexadec-15-ynoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, see Table of Materials) solubilized in chloroform into 1.5 mL tubes.</li>
		<li>Pierce the tubes with a syringe and evaporate the chloroform in a speed-vac at RT for 2 h. Store the dry phospholipid samples at -20 &#176;C.</li>
		<li>Dissolve alkyne-POPE in 0.1% Triton X-100 at 500 &#181;M prior to use. Add a 3 min sonication step in an ultrasonic water bath at RT to solubilize alkyne-POPE if necessary.</li>
		<li>Resuspend FSL-1-biotin in water at 445 &#181;M. 
		NOTE: Both solutions can be stored at -20 &#176;C for at least 2 months.</li>
	</ol>
	</li>
</ol>
2. Tube assay
NOTE: On Day 1, set up the Lnt reaction in 1.5 mL tubes (step 2.1) and coat 96 well plates with streptavidin (step 2.2).
<ol>
	<li>Preparation of reagent mixture and enzymatic reaction
	<ol>
		<li>For a standard Lnt assay, mix alkyne-POPE (final 50 &#181;M from 500 &#181;M stock) and FSL-1-biotin (final concentration of 50 &#181;M from a 445 &#181;M stock) in Lnt reaction buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100 containing 1% BSA) at a final volume of 18 &#181;L in 1.5 mL tubes. Perform all conditions in triplicate.</li>
		<li>Sonicate the substrate mixture (prepared in step 2.1.1) for 3 min in an ultrasonic water bath and incubate at 37 &#176;C for 5 min prior to the addition of Lnt enzyme (step 2.1.3). 
		NOTE: MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate), a thiol-specific reagent, inhibits Lnt8. Add 10 mM MTSES (100 mM stock solution in 100% DMSO) to the reaction tubes (step 2.1.1) to be used as negative control samples. Adjust the volume of the Lnt reaction buffer so the total volume is 18 &#181;L.</li>
		<li>Add active (1 ng/&#181;L, corresponding to 17.2 nM) or inactive Lnt (C387S) (2 &#181;L of 10 ng/&#956;L stock in buffer WA containing 0.05% DDM) and mix with the substrates (step 2.1.2) by pipetting up and down.</li>
		<li>Incubate the reaction at 37 &#176;C for 16 h in a thermomixer with heated lid.</li>
	</ol>
	</li>
	<li>Streptavidin coating of 96 well plates
	<ol>
		<li>Prepare a stock solution of streptavidin at 2 mg/mL in H2O. 
		NOTE: This solution can be stored at -20 &#176;C for up to 6 months.</li>
		<li>From the stock solution prepare 10 &#181;g/mL streptavidin in water as a working solution. Add 100 &#181;L of the solution to each well of the 96 well plate. Bind streptavidin to the plate by incubating at 37 &#176;C overnight without a lid to air-dry the wells.</li>
	</ol>
	</li>
</ol>
NOTE: On Day 2 perform click-chemistry (step 2.3), bind Lnt reaction mixture to streptavidin-coated plates (step 2.4), and detect fluorescence and analyze results (step 2.5).
<ol start="3">
	<li>Click-chemistry reaction
	<ol>
		<li>Prepare stock solutions of Azido-FAM (5 mM in DMSO), TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; 50 mM prepared in water), TBTA (Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; 2 mM in tert-butanol:DMSO (4:1)), and CuSO4&#8729;5H2O (50 mM freshly prepared in H2O).</li>
		<li>In the 1.5 mL tubes containing the Lnt reaction mixture prepared in step 2.1.4, perform click-chemistry by adding the reagents in the following order: 0.2 &#181;L of stock Azido-FAM (final concentration 50 &#181;M), 0.4 &#181;L of stock TCEP (final concentration 1 mM), 0.2 &#181;L of stock TBTA (final concentration 0.02 mM).</li>
		<li>Vortex the solution for 5 s. Then add 0.4 &#181;L of stock CuSO4 (final concentration 1 mM). Vortex again for 5 s. Incubate the samples in the dark at RT for 1 h.</li>
	</ol>
	</li>
	<li>Binding of Lnt reaction mixture to streptavidin-coated plates
	<ol>
		<li>Wash the wells of the streptavidin-coated plates after the overnight incubation from step 2.2.2 3x with 200 &#181;L of PBS-T1 (PBS containing 0.05% Tween-20). 
		NOTE: Streptavidin plates can be used immediately or stored dry at 4 &#176;C.</li>
		<li>Transfer 18 &#181;L from the click-chemistry mixture from step 2.3.3 to a well in a 96 well streptavidin-coated plate and add 100 &#181;L of PBS-T1 to bind N-acyl-FSL-1-biotin-FAM product to streptavidin plates.</li>
		<li>Use biotin-fluorescein (0.26 &#181;M from a 3.1 mM stock in DMSO) as positive control for streptavidin binding and fluorescence readout.</li>
		<li>Incubate the plates at RT for 1 h in the dark in a thermomixer, shaking at 300 rpm.</li>
		<li>Perform manual wash steps of the 96 well plates with a multichannel electronic pipette as follows: six washes with 200 &#181;L of PBS-T2 (PBS containing 1% Tween-20), three flushes with a pipette, three washes with 200 &#181;L of PBS, three flushes with a pipette.</li>
	</ol>
	</li>
	<li>Fluorescence detection and analysis
	<ol>
		<li>Add 200 &#181;L of PBS and record the fluorescence at 520 nm in a fluorescence microplate reader. Save results in spreadsheet software. 
		NOTE: Biotin-fluorescein is used as a positive control for streptavidin binding and fluorescence detection at 520 nm. A reproducible readout of 30,000&#8211;40,000 A.U. with 0.26 &#181;M biotin-fluorescein is expected. All samples are analyzed in triplicate, including controls.</li>
		<li>Calculate the standard deviation for each reaction and the calculate P-values for negative controls and positive samples using unpaired t-test using a statistical software.</li>
	</ol>
	</li>
</ol>
3. Multiwell plate assay
NOTE: On Day 1 set up the Lnt reaction in 384 well plates (step 3.1) and coat 384 well plates with streptavidin (step 3.2).
<ol>
	<li>Preparation of reagent mixture and enzymatic reaction 
	NOTE: The quantity of the reagents and enzyme is reduced compared to the tube assay and the Lnt reaction is performed directly in 384 well plates. This allows the use of less material and a reduction of steps that can be further automated.
	<ol>
		<li>Mix the substrates as follows: alkyne-POPE (final concentration 50 &#181;M from 500 &#181;M stock) and FSL-1-biotin (final 50 &#181;M from 500 &#181;M stock) in Lnt reaction buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100 containing 1% BSA) at a volume of 13.5 &#181;L per reaction per well in a 384 well plate format. Perform all conditions in triplicate. Calculate the total amount of reagents required for the number of reactions to be performed (i.e., 5,184 &#181;L for one 384 well plate).</li>
		<li>Sonicate the substrate mixture for 3 min in an ultrasonic water bath.</li>
		<li>Aliquot 13.5 &#181;L of the substrate mixture per well in a 384 well plate. 
		NOTE: As a negative control, 10 mM of MTSES (625 mM stock solution in 100% DMSO) can be added per well. Adjust the volume of Lnt buffer (step 3.1.1) to reach a final reaction volume of 13.5 &#181;L.</li>
		<li>Incubate at 37 &#176;C for 5 min prior to the addition of Lnt enzyme (step 3.1.5).</li>
		<li>Add 0.5 ng/&#181;L (corresponding to 8.6 nM) active Lnt enzyme, or inactive variant (C387S) (1.5 &#181;L from 5 ng/&#181;L stock in buffer WA containing 0.05% DDM) in Lnt reaction buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 1% BSA) to each well containing the reaction mixture from step 3.1.3. Mix reagents by pipetting up and down. The total reaction volume is 15 &#181;L.</li>
		<li>Seal the plate with plastic foil for multiwell plates.</li>
		<li>Incubate at 37 &#176;C for 16 h in a thermomixer with heated lid.</li>
	</ol>
	</li>
	<li>Streptavidin coating 384 well plates
	<ol>
		<li>Use 75 &#181;L of streptavidin (10 &#181;g/mL in H2O) from step 2.2.2 to coat wells of a 384 well plate. Let streptavidin bind to the plate by incubating at 37 &#176;C overnight without a lid to air-dry the wells.</li>
	</ol>
	</li>
</ol>
NOTE: On Day 2 perform click-chemistry (step 3.3), bind Lnt reaction mixture to streptavidin-coated plates (step 3.4), detect fluorescence, and analyze results (step 3.5).
<ol start="3">
	<li>Click-chemistry reaction
	<ol>
		<li>Prepare stock solutions of Azido-FAM (1.2 mM in DMSO), TCEP (tris(2-carboxyethyl)phosphine hydrochloride; 24 mM prepared in H2O), TBTA (Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; 0.48 mM in tert-butanol:DMSO (4:1)) and CuSO4&#8729;5H2O (24 mM freshly prepared in H2O).</li>
		<li>Combine the three click-chemistry reagents: a mixture of Azido-FAM (final concentration 50 &#181;M), TCEP (1 mM final), and TBTA (0.02 mM final), each at 0.75 &#181;L in final volume of 2.25 &#181;L per well. Calculate the required volume per 384 well plate (i.e., use 864 &#181;L when all wells of the 384 well plate are used). Mix vigorously.</li>
		<li>Add 2.25 &#181;L of the reagent solution per well directly to the completed Lnt reaction in the 384 well plate from step 3.1.7.</li>
		<li>Mix by pipetting up and down with a multichannel electronic pipette.</li>
		<li>Add CuSO4 (0.75 &#181;L from 24 mM stock for a 1 mM final concentration) and mix by pipetting up and down with a multichannel electronic pipette.</li>
		<li>Incubate at RT for 1 h in the dark.</li>
	</ol>
	</li>
	<li>Binding of Lnt reaction mixture to streptavidin-coated 384 well plates
	<ol>
		<li>Wash the wells of the streptavidin-coated plates after the overnight incubation from step 3.2.1 1x with 85 &#181;L PBS-T1.</li>
		<li>Transfer 11 &#181;L of the click-chemistry reaction from each well of the incubation plate from step 3.3.6 to the streptavidin-coated plate from step 3.4.1 to bind the N-acyl-FSL-1-biotin-FAM product to the streptavidin plates.</li>
		<li>Add 64 &#181;L of buffer PBS-T1.</li>
		<li>Include biotin-fluorescein (0.19 &#181;M from a 3.1 mM stock in DMSO) as a control for binding to streptavidin-coated wells.</li>
		<li>Incubate the 384 well plates at RT for 1 h in the dark in a thermomixer, shaking at 300 rpm.</li>
		<li>Wash the plates using an automated plate washer as follows: 10 washes with 85 &#181;L PBS-T2, plates are shaken between washes; 5 washes with 85 &#181;L PBS, plates are shaken between washes.</li>
	</ol>
	</li>
	<li>Fluorescence detection and analysis
	<ol>
		<li>Add PBS (85 &#181;L) and record fluorescence in a fluorescence microplate reader at 535 nm.</li>
		<li>Analyze data as described (step 2.5.2).</li>
	</ol>
	</li>
</ol>

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	<li>Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+bacterial+lipoprotein+modification--how,+when+and+why.">The molecular mechanism of bacterial lipoprotein modification--how, when and why.</a> FEMS Microbiology Reviews. 39 (2), 246-261 (2015).</li><li>Kovacs-Simon, A., Titball, R. W., Michell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+bacterial+pathogens.">Lipoproteins of bacterial pathogens.</a> Infections and Immunity. 79 (2), 548-561 (2010).</li><li>Jackowski, S., Rock, C. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Visualization+and+Large-Scale+Profiling+of+Bacterial+Lipoproteins+with+Chemical+Reporters.">Rapid Visualization and Large-Scale Profiling of Bacterial Lipoproteins with Chemical Reporters.</a> Journal of American Chemical Society. 132 (31), 10628-10629 (2010).</li><li>Thiele, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+fatty+acid+metabolism+by+click-chemistry.">Tracing fatty acid metabolism by click-chemistry.</a> ACS Chemical Biology. 7 (12), 2004-2011 (2012).</li><li>Ganesan, L., Shieh, P., Bertozzi, C. R., Levental, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-Chemistry+Based+High+Throughput+Screening+Platform+for+Modulators+of+Ras+Palmitoylation.">Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation.</a> Science Reports. 7, 41147 (2017).</li><li>Nozeret, K., Boucharlat, A., Agou, F., Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+fluorescence-based+assay+to+monitor+enzymatic+activity+of+the+essential+integral+membrane+protein+Apolipoprotein+N-acyltransferase+(Lnt).">A sensitive fluorescence-based assay to monitor enzymatic activity of the essential integral membrane protein Apolipoprotein N-acyltransferase (Lnt).</a> Science Reports. 9 (1), 15978 (2019).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecules Screening. 4 (2), 67-73 (1999).</li><li>Mao, 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=Crystal+structure+of+E.+coli+lipoprotein+diacylglyceryl+transferase.">Crystal structure of E. coli lipoprotein diacylglyceryl transferase.</a> Nature Communication. 7, 10198 (2016).</li><li>Vogeley, L., 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=Structural+basis+of+lipoprotein+signal+peptidase+II+action+and+inhibition+by+the+antibiotic+globomycin.">Structural basis of lipoprotein signal peptidase II action and inhibition by the antibiotic globomycin.</a> Science. 351 (6275), 876-880 (2016).</li><li>Wiktor, 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=Structural+insights+into+the+mechanism+of+the+membrane+integral+N-acyltransferase+step+in+bacterial+lipoprotein+synthesis.">Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.</a> Nature Communication. 8, 15952 (2017).</li><li>Noland, C. L., 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=Structural+insights+into+lipoprotein+N-acylation+by+Escherichia+coli+apolipoprotein+N-acyltransferase.">Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase.</a> Proceedings of the National Academy of Sciences USA. 114 (30), 6044-6053 (2017).</li><li>Lu, 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=Crystal+structure+of+E.+coli+apolipoprotein+N-acyl+transferase.">Crystal structure of E. coli apolipoprotein N-acyl transferase.</a> Nature Communication. 8, 15948 (2017).</li></ol>

The authors have nothing to disclose.

The protocol for the Lnt assay described here, based on fluorescence detection of the triacylated product, is sensitive and reproducible. The specific and efficient binding of biotin to streptavidin is a key element in the assay. Alkyne-POPE substrate left after completion of the Lnt reaction is also fluorescently labeled with FAM but is efficiently removed after binding onto the streptavidin plates by multiple wash steps. Furthermore, addition of DMSO does not affect Lnt activity and has no impact on the assay. Both sub...

Bacterial lipoproteins are characterized by covalently bound fatty acids at their amino-termini through which they are anchored into membranes1,2. The mature part of the protein is highly diverse in structure and function, thereby explaining the role of lipoproteins in various biological processes in the bacterial cell envelope.
Lipoproteins are modified by phospholipid-derived fatty acids after insertion into the cytoplasmic membrane. The prolipoproteins contain a signature motif, the lipobox, which contains an invariant cysteine residue that becomes acylated and the first amino acid in the mature protein. The first step of this pathway is catalyzed by prolipoprotein phosphatidylglycerol::diacylglyceryl transferase (Lgt), which transfers the diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein via a thioether link between diacylglyceryl and cysteine. Signal peptidase II (Lsp) cleaves the signal peptide from diacylglyceryl prolipoprotein, resulting in an apolipoprotein that is anchored into the membrane through its diacylglyceryl moiety. The third and last step is catalyzed by apolipoprotein N-acyltransferase (Lnt), which adds a fatty acid from the sn-1 position of phospholipid onto apolipoprotein, resulting in triacylated mature lipoprotein (Figure 1)3. The Lnt reaction is a two-step ping-pong reaction where a stable thioester acyl enzyme intermediate is formed. The lysophospholipid byproduct is released prior to the acylation of the apolipoprotein substrate in the second step of the reaction.
The phospholipid substrate specificity is determined in a Lnt assay based on the mobility shift of N-acyl diacylglyceryl peptide on a high percentage Tris-Tricine Urea SDS-PAGE4. Phospholipids with small polar headgroups, saturated [sn-1] and nonsaturated [sn-2], were preferred substrates4. The gel shift assay is not appropriate for extensive kinetic studies of apolipoprotein N-acyltransferase nor for HTS to identify inhibitory molecules. Click-chemistry using alkyne fatty acids has been successfully used to study lipoprotein modification in bacteria5 and fatty acid metabolism in eukaryotes6. Recently, an in vitro assay of Ras palmitoylation was reported to identify inhibitors7.
In the method described here, purified active Lnt in detergent is incubated with substrates in mixed micelles to form alkyne-triacylated peptide that is subsequently detected by fluorescence spectrometry.

<table>
	<tbody>
		<tr>
			<td>&#196;kta Purifier FPLC system</td>
			<td>GE Healthcare</td>
			<td>NA</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>alkyne-POPE</td>
			<td>Avanti Polar Lipids</td>
			<td>900414P</td>
			<td>Purity: &#62;99% 
			Lnt substrate</td>
		</tr>
		<tr>
			<td>Azido-FAM</td>
			<td>Lumiprobe</td>
			<td>A4130</td>
			<td>Purity: 100% (pure) 
			Click reagent</td>
		</tr>
		<tr>
			<td>BioPhotometer Plus</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td>Purity: NA 
			OD 600 nm</td>
		</tr>
		<tr>
			<td>BioTek ELX 405 Select plate washer</td>
			<td>BioTek</td>
			<td>N&#176; serie 115800, n&#176; materiel 405 Select</td>
			<td>Purity: NA 
			Wash steps</td>
		</tr>
		<tr>
			<td>biotin-fluorescein</td>
			<td>Sigma</td>
			<td>53608</td>
			<td>Purity: &#8805;90% 
			Fluorescence control</td>
		</tr>
		<tr>
			<td>Copper(II) sulfate pentahydrate</td>
			<td>Sigma</td>
			<td>C3036</td>
			<td>Purity: &#8805;98% 
			Click reagent</td>
		</tr>
		<tr>
			<td>DDM</td>
			<td>Anatrace</td>
			<td>D310A</td>
			<td>Purity: &#8805; 99% &#946;+&#945;; &#60; 15% &#945; 
			Detergent for Lnt purification</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Invitrogen</td>
			<td>D12435</td>
			<td>Purity: anhydrous 
			Solbilization Click reagent</td>
		</tr>
		<tr>
			<td>Electronic pipet Voyager 8 channels 0.5-12.5 uL</td>
			<td>INTEGRA</td>
			<td>4721</td>
			<td>Purity: NA 
			Handling reagents</td>
		</tr>
		<tr>
			<td>French Pressure Cell</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purity: NA 
			Cell disruption</td>
		</tr>
		<tr>
			<td>FSL-1-biotin</td>
			<td>EMC microcollections</td>
			<td>L7030</td>
			<td>Purity: NA 
			Lnt substrate</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 384-well standard CELLSTAR polystyrene microplate</td>
			<td>Greiner</td>
			<td>781091</td>
			<td>Purity: NA 
			Black with transparent bottom (up or bottom reading)</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 96-well sterile polystyrene plate, high binding</td>
			<td>Greiner</td>
			<td>655097</td>
			<td>Purity: NA 
			Black with transparent bottom (up or bottom reading)</td>
		</tr>
		<tr>
			<td>Microplate reader Infinite M1000 pro</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate)</td>
			<td>Anatrace</td>
			<td>S110MT</td>
			<td>Purity: ~100% 
			Thiol specific inhibitor</td>
		</tr>
		<tr>
			<td>Optically Clear Adhesive Seal Sheets</td>
			<td>Thermo Scientific</td>
			<td>AB-1170</td>
			<td>Purity: NA 
			Foil to seal multi-well plate</td>
		</tr>
		<tr>
			<td>Sephacryl S400 HR 16/60 gel filtration column</td>
			<td>GE Healthcare</td>
			<td>GE28-9356-04</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>StrepTactin Sepharose 50 %</td>
			<td>IBA Biotechnology</td>
			<td>2-1201-010</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>streptavidin</td>
			<td>Sigma</td>
			<td>S4762-1MG</td>
			<td>Purity: &#8805;13 units/mg protein 
			Biotin binding</td>
		</tr>
		<tr>
			<td>TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine</td>
			<td>Sigma</td>
			<td>678937</td>
			<td>Purity: 97% 
			Click reagent</td>
		</tr>
		<tr>
			<td>TCEP (tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Sigma</td>
			<td>75259</td>
			<td>Purity: &#8805;98% 
			Click reagent</td>
		</tr>
		<tr>
			<td>TECAN Infinite F500</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>TECAN Infinite M1000 pro</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>Thermomixer C</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td>Purity: NA 
			Heated lid</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>93443</td>
			<td>Purity: 10% in H2O 
			Lnt reaction buffer</td>
		</tr>
		<tr>
			<td>Ultra centrifuge</td>
			<td>Beckman LC</td>
			<td>N/A</td>
			<td>Purity: NA 
			Cell fractionation</td>
		</tr>
	</tbody>
</table>

click-chemistry based fluorometric assay for apolipoprotein n-acyltransferase from enzyme characterization to high-throughput screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

In the Lnt reaction the sn-1 fatty acid from phospholipids is transferred onto a diacylglyceryl peptide, resulting in mature triacylated peptide8. The in vitro Lnt assay described here is designed to use phospholipids containing an alkyne fatty acid (alkyne-POPE) and FSL-1-biotin as substrates, resulting in the formation of alkyne-FSL-1-biotin. Upon a click-chemistry reaction with azido-FAM, this product should become fluorescently labeled and detected by fluorescence spectrometry (<stron...

Watch this Scientific Journal Video about Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening at JoVE.com

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

click-chemistry-based-fluorometric-assay-for-apolipoprotein-n

Research

JoVE Journal

Biochemistry

1.8K Views.  Institut Pasteur, Unité Biologie et génétique de la paroi bactérienne, Paris, France ; CNRS, UMR 2001 « Microbiologie intégrative et Moléculaire », Paris, France ; INSERM, Équipe Avenir, Paris, France. Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Video: Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

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 not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

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

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...