Name: utilizing the ethylene-releasing compound, 2-chloroethylphosphonic acid, as a tool to study ethylene response in bacteria
Uploaded: 2016-11-10T14:00:00
Description: Watch this Scientific Journal Video about Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria at JoVE.com

The authors thank Dr. Dario Bonetta for providing Arabidopsis thaliana seeds and for technical assistance in regards to the triple response assay, as well as Simone Quaranta for help with FT-IR. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC-DG) to JLS, an Ontario Graduate Scholarship (OGS) to RVA, and a Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST) to AJV.

1. Chemicals
<ol>
	<li>Prepare a solution of 500 mM CEPA (144.49 g/mol), and a solution consisting of both 500 mM NaCl (58.44 g/mol) and 500 mM NaH2PO4&#183;H2O (137.99 g/mol) in acidified (pH 2.5) ultra-pure water or 0.1 N HCl. Mix using a vortex until the solutions are clear.</li>
	<li>Serially dilute (10x) the 500 mM solutions in the same solvent to obtain 5 mM and 50 mM stocks.</li>
	<li>Prepare a 10 mM solution of 1-aminocyclopropane carboxylic acid (ACC; 101.1 g/mol) in ultra-pure...

<ol>
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Sci. 88 (16), 7434-7437 (1991).</li><li>Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+ethylene+removal+with+Pseudomonas+strains.">Assessment of ethylene removal with Pseudomonas strains.</a> J. Hazard. Mater. 131 (3), 131-136 (2006).</li><li>Kim, H. E., Shitashiro, M., Kuroda, A., Takiguchi, N., Kato, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethylene+chemotaxis+in+Pseudomonas+aeruginosa+and+other+Pseudomonas+species.">Ethylene chemotaxis in Pseudomonas aeruginosa and other Pseudomonas species.</a> Microbes Environ. 22 (2), 186-189 (2007).</li><li>Augimeri, R. V., Strap, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phytohormone+ethylene+enhances+bacterial+cellulose+production,+regulates+CRP/FNRKx+transcription+and+causes+differential+gene+expression+within+the+cellulose+synthesis+operon+of+Komagataeibacter+(Gluconacetobacter)+xylinus+ATCC+53582.">The phytohormone ethylene enhances bacterial cellulose production, regulates CRP/FNRKx transcription and causes differential gene expression within the cellulose synthesis operon of Komagataeibacter (Gluconacetobacter) xylinus ATCC 53582.</a> Front. Microbiol. 6, 1459 (2015).</li><li>Zhang, W., Wen, C. 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The methods described here outline the in situ production of ethylene from CEPA for the study of bacterial ethylene response using the model organism, K. xylinus. This method is very useful as ethylene can be produced by supplementing any aqueous medium that has a pH greater than 3.510,11 with CEPA negating the need for pure ethylene gas or specialized laboratory equipment. This method is not limited to studying the effects of CEPA-derived ethylene on bacteria but can be also be adapted to st...

The olefin ethylene (C2H4) was first discovered as a plant hormone in 1901 when it was observed that pea seedlings, grown in a laboratory that used coal gas lamps, exhibited an abnormal morphology in which stems (hypocotyls) were shorter, thicker and bent sideways compared to normal pea seedlings; a phenotype later termed the triple response1,2. Subsequent studies demonstrated that ethylene is a vital phytohormone that regulates numerous developmental processes such as growth, stress response, fruit ripening and senescence3. Arabidopsis thaliana, a model organism for plant biology research, has been well studied in regards to its response to ethylene. Several ethylene response mutants have been isolated by exploiting the triple response phenotype observed in dark-grown A. thaliana seedlings in the presence of ethylene1,4,5. The biosynthetic precursor for ethylene production in plants is 1-aminocyclopropane carboxylic acid (ACC)6 and is commonly used during the triple response assay to increase endogenous ethylene production that leads to the triple response phenotype1,4,5.
Although the ethylene response is widely studied in plants, the effect of exogenous ethylene on bacteria is vastly understudied despite the close association of bacteria with plants. One study reported that certain Pseudomonas strains can survive using ethylene as a sole source of carbon and energy7. However, only two studies have demonstrated that bacteria respond to ethylene. The first study showed that strains of Pseudomonas aeruginosa, P. fluorescens, P. putida, and P. syringae were chemotactic toward ethylene using an agarose plug assay in which molten agarose was mixed with a chemotaxis buffer equilibrated with pure ethylene gas8. However, to our knowledge, there have been no further reports using pure ethylene gas to characterize bacterial ethylene response, likely due to the difficultly of handling gases in the laboratory without specialized equipment. The second report of bacterial ethylene response demonstrated that ethylene increased bacterial cellulose production and influenced gene expression in the fruit-associated bacterium, Komagataeibacter (formerly Gluconacetobacter) xylinus9. In this case, the ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA) was used to produce ethylene in situ within the bacterial growth medium, bypassing the need for pure ethylene gas or specialized equipment.
CEPA produces ethylene at a 1:1 molar ratio above pH 3.510,11 through a base-catalyzed, first-order reaction12-14. The degradation of CEPA is positively correlated with pH and temperature13,14 and results in the production of ethylene, chloride and phosphate. CEPA provides researchers interested in studying bacterial responses to ethylene with a convenient alternative to gaseous ethylene.
The overall goal of the following protocols is to provide a simple and efficient method to study bacterial ethylene response and includes validation of physiologically relevant levels of ethylene production from CEPA decomposition in bacterial growth medium, analysis of culture pH to ensure CEPA decomposition is not impaired during bacterial growth, and assessment of the effect of ethylene on bacterial morphology and phenotype. We demonstrate these protocols using K. xylinus, however, these protocols can be adapted to study ethylene response in other bacteria by using the appropriate growth medium and phenotype analyses.

<table border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">1-aminocyclopropane carboxylic acid (ACC)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">A3903</td>
			<td style="width:432px;">Biosynthetic precursor of ethylene in plants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">4-sector Petri dish</td>
			<td style="width:136px;">Phoenix Biomedical</td>
			<td style="width:119px;">CA73370-022</td>
			<td>For testing triple response</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Agar</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">AGR001.1</td>
			<td>To solidify medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Canon Rebel T1i DLSR camera</td>
			<td style="width:136px;">Canon</td>
			<td style="width:119px;">3818B004</td>
			<td>For pictures of pellicles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Cellulase from Trichoderma reesei ATCC 26921&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C2730</td>
			<td>Aqueous solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Citric acid</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">CIT002.500</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Commercial bleach</td>
			<td style="width:136px;">Life Brand</td>
			<td style="width:119px;">57800861874</td>
			<td>Bleach for seed sterilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Concentrated HCl</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">HCL666.500</td>
			<td>Hydrochloric acid for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital USB microscope</td>
			<td style="width:136px;">Plugable</td>
			<td style="width:119px;">N/A</td>
			<td>For pictures of colonies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ethephon (&#8805; 96%; 2-chloroethylphosphonic acid)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C0143</td>
			<td>Ethylene-releasing compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glucose</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">GB0219</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Komagataeibacter xylinus ATCC 53582</td>
			<td style="width:136px;">ATCC</td>
			<td style="width:119px;">53582</td>
			<td>Bacterial cellulose-producing alphaproteobacterium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microcentrifuge tube</td>
			<td style="width:136px;">LifeGene</td>
			<td style="width:119px;">LMCT1.7B</td>
			<td>1.7 mL microcentrifuge tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Murashige and Skoog (MS) basal medium&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">M5519</td>
			<td>Arabidopsis thaliana growth medium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Na2HPO4&#183;7H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPD579.500</td>
			<td>Sodium phosphate, dibasic heptahydrate for SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaCl</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">SOD001.1</td>
			<td>Sodium chloride for saline and control solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">NaH2PO4&#183;H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPM306.500</td>
			<td>Sodium phosphate, monobasic monohydrate for control solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaOH</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SHY700.500</td>
			<td>Sodium hydroxide for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Paraffin film</td>
			<td style="width:136px;">Parafilm</td>
			<td style="width:119px;">PM996</td>
			<td>For sealing plates and flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Peptone (bacteriological)</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">PEP403.1</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Petroff-Hausser counting chamber</td>
			<td style="width:136px;">Hausser scientific</td>
			<td style="width:119px;">3900</td>
			<td>Bacterial cell counting chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Polyethersulfone sterilization filter 0.2 &#181;m</td>
			<td style="width:136px;">VWR</td>
			<td style="width:119px;">28145-501</td>
			<td>For sterilizing cellulase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sucrose</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SUC600.1</td>
			<td>Sucrose for MS medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Yeast extract</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">G0961</td>
			<td>For SH medium</td>
		</tr>
	</tbody>
</table>

utilizing the ethylene-releasing compound, 2-chloroethylphosphonic acid, as a tool to study ethylene response in bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

A schematic plate setup for verification of ethylene liberation from CEPA in SH medium (pH 7) by the triple response assay is shown in Figure 1A-C. A flow-chart illustrating the pellicle protocol is shown in Figure 2. Dark-grown A. thaliana seedlings exhibit the triple response phenotype (shorter and thicker hypocotyl with an exaggerated apical hook) in the presence of ACC and in the presence of ethylene produced through the dec...

Watch this Scientific Journal Video about Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria at JoVE.com

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

The overall goal of this procedure is to provide a simple and safe method for the study of bacterial response to ethylene. This method will answer key questions regarding plant-microbe interactions, such as the role that ethylene plays in bacterial adhesion and colonization of plants. The main advantage of this technique is that it does not require any specialized equipment and it's much safer than handling gaseous ethylene in the lab.Although this method can provide insight into how bacterial cellulose production is affected by ethylene, it can also be applied to other plant-associated microorganisms for the in vitro study of how ethylene-mediated processes are involved in plant colonization. In preparation, make 100 milliliters of growth medium for the Arabidopsis seedlings and make 100 milliliters of bacterial growth medium for the CEPA decomposition. Once the arabidopsis medium has tempered, divide 40 milliliters out to a sterile flask and supplement the aliquot with 40 microliters of 10 millimolar ACC.Now, load the plate quadrants with five milliliters of medium and allow the agar to solidify. Opposing quadrants of all plates have bacterial growth medium and arabidopsis medium. Use three different medium configurations.The CEPA treatment will occur later in the protocol. Prepare each plate configuration in triplicate. Once the agar has cooled, evenly distribute approximately 50 seeds onto each quadrant of arabidopsis medium.Then, incubate the plates in the dark at four degrees Celsius for three to four days. Later, after incubating the seeds, expose them to fluorescent light for two hours. Then, spread 10 microliters of 500 millimolar CEPA stock solution onto the bacterial agar of one plate configuration.The final CEPA concentration in the agar will be one millimolar. Now, seal all the plates with laboratory film and wrap them in foil. Then, place the plates in a dark environment at 23 degrees Celsius with the agar side down and let them incubate for three days to germinate the seeds.After the seeds germinate, use flame sterilized forceps to remove seedlings corresponding to each treatment. Select 30 seedlings from each quadrant for a total of 180 per treatment. Image the seedlings under a dissecting microscope or with a high resolution digital camera against a black background next to a ruler for scale.Then, measure their hypocotyl length using ImageJ software. Using the straight tool, measure 10 millimeters and select Set Scale under the Analyze tab to set the scale. Then, use the Segmented tool under the Straight Line tool to measure each hypocotyl and press M to calculate their lengths.The pellicle assay is a standard analysis tool for bacterial cellulose production. PH and morphological analysis are detailed in sex protocol. In preparation, harvest cells from starter cultures in triplicate and quantify the cells using a Petroff-Hauser Counting Chamber.Then, prepare four medium master mixes with different CEPA concentrations. For each master mix, eliquate 60 milliliters of pH 7 SH medium and add 120 microliters of either zero, five, 50 or 500 millimolar CEPA stock solution to obtain final CEPA concentrations of zero, 01, 0.1, or 1.0 millimolar. Mix these media by swirling the flasks.One set of media is needed for each of the triplicates and a fourth set is needed to function as a sterile control. So, divide each 60 milliliter medium preparation into four 14 milliliter eliquates. Then, pre-cool the 14 milliliter eliquates before inoculating with their respective starter cultures for a final concentration of 100, 000 cells per milliliter.Keep all the inoculated tubes on ice to prevent cellulose production. Use the remaining 14 milliliter eliquates for sterile control wells. Now, from each 14 milliliter master mix, load two milliliter eliquates into six wells of a sterile 24 well plate for a total of four loaded plates per starter culture analyzed.Carefully seal the plates with paraffin film and incubate them statically for seven days at 30 degrees Celsius. During the incubation, incubate a 25 milliliter liquid culture in parallel to periodically measure the medium's acidity. If the acidity drops below a PH of five, then the organism's growth will inhibit the decomposition of CEPA to ethylene and is thus incompatible with this assay.A week later, harvest and measure pellicle wet weight, thickness, dry weight and crystallinity. To collect a pellicle from the plate, depress one side of the pellicle to elevate the opposing edge and pick it up with forceps. While retaining grip, place them on a fresh paper towel for three seconds to remove excess medium.Then, weigh each pellicle and record this as the wet weight. Next, align the pellicles along a ruler and photograph them from the side using a high resolution digital camera. Analyze these photos for the pellicles'thicknesses.Next, individually transfer the pellicles into the wells of a six well plate. Treat them with 12 milliliters of 0.1 normal sodium hydroxide at 80 degrees Celsius for 20 minutes to lice the cells. Then, remove the sodium hydroxide and neutralize the treated pellicles by thoroughly washing them with 12 milliliters of ultra pure water for 24 hours with agitation in a six well plate.During this incubation, change the water every six hours. When the incubation is over, the pellicles should be white. Then, place the pellicles on silicon mats and dry them at 50 degrees Celsius for 48 hours to prepare them for dry weight measurement.The pellicles'crystallinity can be analyzed using Fourier-transform infrared spectroscopy. Dark grown arabidopsis thaliana seedlings exhibited the triple response phenotype of a short, thick hypocotyl with an exaggerated apical hook in the presence of ACC and in the presence of ethylene produced through the decomposition CEPA on SH medium, but not under untreated conditions. Clearly, the hypocotyl length of the treated seeds was significantly less than the untreated controls.Thus, ethylene was released from CEPA on SH medium at a physiologically relevant concentration. All concentrations of CEPA derived ethylene significantly decreased pellicle wet weight, but did not affect pellicle thickness, and all concentrations of CEPA derived ethylene significantly increased pellicle dry weight. Furthermore, pellicle hydration was reduced by all concentrations of CEPA derived ethylene.While attempting this procedure, it's important to measure the PH of the growth medium to ensure that the PH does not drop below five so that CEPA degradation into ethylene is not inhibited. Remember that powdered CEPA is extremely hazardous to mucous membranes. Proper care should always be taken when performing this protocol.This technique will enable researchers who are interested in plant bacteria interactions to safely explore ethylene mediated responses in vitro. This will help shed light on how plant signals mediate bacterial colonization and adhesion. Currently, this area has been under studied.

utilizing-ethylene-releasing-compound-2-chloroethylphosphonic-acid-as

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

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 ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

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

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

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

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

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...