This work was partially funded by FAI-UASLP grant number C19-FAI-05-89.89 and CONACYT grant number 316463 (Apoyos a la Ciencia de Frontera: Fortalecimiento y Mantenimiento de Infraestructuras de Investigaci&#243;n de Uso Com&#250;n y Capacitaci&#243;n T&#233;cnica 2021). The authors thank the technical assistance of Julian E. Mata-Morales in video edition.

1. Determination (in silico) of the ANS and SERCA N-domain interaction
<ol>
	<li>Generate a three-dimensional (3D) structure of the protein (SERCA N-domain) by molecular modeling using the preferred protein modeling software50.</li>
	<li>Identify the amino acid residues that form the nucleotide-binding site using the preferred molecular structure software51, and determine the presence of Arg and Lys residues35; these are required for ANS binding and to increase the fluorescence intensity (quantum yield).</li>
	<li>Perform molecular docking (using the preferred docking software)52,53,54 to determine the interactions of ATP, fluorescein isothiocyanate (FITC) (which forms a covalent bond with Lys515 labeling the nucleotide-binding site), and ANS with amino acids residues in the nucleotide-binding site (Figure 1).</li>
	<li>Calculate the molecular distance (&#197;) between Trp residue and bound ANS using the measurement tool in the preferred software.</li>
	<li>Perform molecular dynamics simulation of ANS-N-domain complex to determine the stability of the interaction52,54. Then, perform the in vitro experiments when the stability of the complex has been confirmed.</li>
</ol>
2. Expression and purification of the recombinant N-domain
<ol>
	<li>Synthesize the gene coding for N-domain40.</li>
	<li>Design and construct the plasmid that contains the synthetic gene that codes for the N-domain40.</li>
	<li>Express and purify by affinity chromatography (Ni-NTA), the engineered recombinant N-domain. Perform an SDS-PAGE of the purified protein to determine the purity (Figure 2)40.</li>
	<li>Determine the protein concentration by studying the absorbance at &#955; of 280 nm with the N-domain extinction coefficient (&#949;= 11,960 M-1&#183;cm-1)40.</li>
</ol>
3. Monitor the formation of the ANS-N-domain complex based on ANS and N-domain fluorescence intensity changes.
<ol>
	<li>Prepare an ANS stock solution in N,N-dimethylformamide.
	<ol>
		<li>Weigh a small amount (1-5 mg) of ANS, and dissolve it in 1 mL of the final volume of N,N-dimethylformamide, e. g., 3.2 mg (10.69 mM final concentration).</li>
		<li>Prepare a 100 &#181;M ANS aqueous stock solution using the ANS solution in N,N-dimethylformamide, e. g., add 9.4 &#181;L of the 10.69 mM ANS solution to 990.6 &#181;L of 50 mM phosphate buffer with pH 8.0 to obtain a final volume of 1 mL.</li>
		<li>Mix the solutions by vortexing 3 - 5 times for 15 s. 
		NOTE: In the following experiment, use only the ANS aqueous stock solution. Freshly prepare the ANS aqueous stock solution before initiating the experiments.</li>
	</ol>
	</li>
	<li>Prepare the NBS stock solution in N,N-dimethylformamide.
	<ol>
		<li>Weigh a small amount (1-5 mg) of NBS, and dissolve it in 1 mL of N,N-dimethylformamide, e. g., 5.3 mg in 1 mL (29.78 mM final concentration).</li>
		<li>Prepare a 1 mM NBS aqueous stock solution using the NBS solution in N,N-dimethylformamide, e. g., add 3.36 &#181;L of the 29.78 mM NBS solution to 96.64 &#181;L of 50 mM phosphate buffer with pH 8.0 to obtain a final volume 0.1 mL.</li>
		<li>Mix the solutions by vortexing 3 - 5 times for 15 s. 
		NOTE: Freshly prepare the NBS aqueous stock solution before starting the experiments.</li>
	</ol>
	</li>
	<li>Titrate the N-domain with ANS, and record the fluorescence spectra by excitation at &#955;=295 nm at 25 &#176;C.
	<ol>
		<li>Obtain the fluorescence spectrum baseline.
		<ol>
			<li>Place 1 mL of 50 mM phosphate buffer with pH 8.0 in a 1 mL fluorescence quartz cuvette.</li>
			<li>Position the cell in the thermo-stated cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
			<li>Record the fluorescence spectrum (305 - 550 nm). 
			NOTE: The fluorescence spectrum of the 50 mM phosphate buffer with pH 8.0, which serves as the blank sample, is subtracted from all obtained fluorescence spectra.</li>
		</ol>
		</li>
		<li>Obtain the intrinsic fluorescence spectrum of the N-domain.
		<ol>
			<li>Place 900 &#181;L of 50 mM phosphate buffer with pH 8.0 in a fluorescence quartz cuvette.</li>
			<li>Add 100 &#181;L of N-domain (10 &#956;M) suspension to obtain a 1 &#956;M N-domain final concentration in a 1 mL final volume.</li>
			<li>Gently homogenize using a micropipette 20 times to ensure the homogeneity of the solution. 
			NOTE: The protein should be freshly purified to obtain high-quality intrinsic fluorescence spectra, e. g., the purified recombinant N-domain may only be used for a week after purification.</li>
			<li>Position the cell in the thermo-stated cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
			<li>Record the N-domain intrinsic fluorescence spectrum (305-550 nm).</li>
		</ol>
		</li>
		<li>Add ANS, and obtain the fluorescence spectrum by excitation at &#955;=295 nm.
		<ol>
			<li>Add a 2 &#181;L aliquot of 100 &#181;M ANS aqueous stock solution to the suspended N-domain (1 &#956;M) to obtain a 0.2 &#181;M ANS final concentration.</li>
			<li>Gently homogenize using a micropipette 20 times to ensure the homogeneity of the solution.</li>
			<li>Position the cell in the thermo-stable cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
			<li>Record the fluorescence spectrum (305-550 nm).</li>
			<li>Repeat the ANS additions and fluorescence spectra recording above 1:1 molar relationship ANS:N-domain.</li>
			<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
			<li>Plot all the spectra in a single graph.</li>
			<li>Determine whether the spectra form a FRET-like pattern. The ANS-N-domain fluorescence spectra form a FRET-like pattern (Figure 3A).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. N-domain intrinsic fluorescence titration by Trp chemical modification with NBS.
<ol>
	<li>Repeat steps 3.3.1 and 3.3.2.</li>
	<li>Add a 1 &#181;L aliquot of 1 mM NBS aqueous stock solution to the suspended N-domain (1 &#956;M) to obtain a final concentration of 1 &#181;M NBS.</li>
	<li>Gently homogenize by using a micropipette 20 times to ensure the homogeneity of the solution.</li>
	<li>Position the cell in the thermo-stable cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
	<li>Record the fluorescence spectrum (305-550 nm) (Figure 3B).</li>
	<li>Repeat the NBS addition and fluorescence spectra recording until minimal N-domain intrinsic fluorescence quenching is observed40. In the N-domain, this usually occurs at a molar ratio of 5-6 NBS/N-domain40. 
	NOTE: NBS rapidly quenches (&#60;5 s) the intrinsic fluorescence of the N-domain; a decrease in fluorescence intensity is observed. Proceed immediately to the next step, as NBS may also react with other amino acid residues47.</li>
	<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
	<li>Plot all spectra in a single graph (Figure 3B).</li>
</ol>
5. Titrate the NBS-modified N-domain with ANS by recording fluorescence spectra at 25 &#176;C.
<ol>
	<li>Perform Step 3.3.3 using the NBS modified N-domain that was generated in Step 4.</li>
	<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
	<li>Plot all spectra in a single graph (Figure 3C).</li>
	<li>The generated fluorescence spectra (Figure 3C) support or refute the occurrence of FRET, i.e., when FRET occurs, the ANS fluorescence does not increase and vice-versa.</li>
</ol>
6. Evidence of ANS binding to the chemically modified N-domain by excitation at &#955;=370 nm.
<ol>
	<li>Perform Step 3.3.3 using the NBS modified N-domain that was generated in Step 4 but changing the excitation &#955; to 370 nm.</li>
	<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
	<li>Plot all spectra in a single graph (Figure 3D).</li>
	<li>Confirm ANS binding to the N-domain by observing the increase in ANS fluorescence intensity. ANS binding to the N-domain shows a fluorescence increase when excited at &#955;=370 nm (Figure 3D). As a control, the fluorescence spectrum of ANS (alone) in 50 mM phosphate buffer with pH 8.0 was obtained exciting at &#955; of 295 and 370 nm (Figure 4, not shown in video). 
	NOTE: The stoichiometric relationship of NBS:Trp that is required for chemical modification depends on the degree of burying of the Trp residue(s) in the protein under study46,47,55,56. Therefore, it is recommended to determine the NBS:protein/(Trp) molar ratio, beforehand.</li>
</ol>

The authors declare that they have no competing financial interests.

Fluorescence spectra of the ANS-N-domain complex display a FRET-like pattern when excited at a &#955; of 295 nm, while the molecular distance (20 &#197;) between the Trp residue and ANS seems to support the occurrence of FRET (Figure 1). Trp chemical modification by NBS results in a less fluorescent N-domain (Figure 3B, Spectrum f); hence, energy transfer is not possible. The ANS fluorescence spectra are similar to that of the nonmodified N-domain when excited a...

F&#246;ster resonance energy transfer (FRET) has become a standard technique for determining the distance between molecular structures after binding or interaction in protein structure and function studies1,2,3,4. In P-type ATPases, FRET has been used to investigate the structure and function of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)2,5,6,7,8, e. g., structural fluctuations during the catalytic cycle have been analyzed in the whole protein by FRET7.
FRET donors are diverse, and range from small fluorescent (extrinsic) molecules to fluorescent proteins9,10. Tryptophan (Trp) residues (due to their fluorescence) are useful for identifying structural changes in protein amino acid sequences11,12. The fluorescence intensity of Trp depends substantially on the polarity of its surrounding environment13,14. Ligand binding usually generates structural rearrangements in proteins/enzymes15,16. If Trp is present at or located close to the protein binding site, structural fluctuations frequently affect the degree of Trp exposure to aqueous media13,14; thus, the change in polarity results in quenching of the Trp fluorescence intensity13,14. Hence, the fluorescent property of Trp is useful for performing ligand binding studies for enzymes. Other physical phenomena may also lead to Trp fluorescence quenching17,18,19,20, e. g., FRET and changes in medium polarity. Energy transfer from the excited state of Trp to a fluorophore also has potential applications, e. g., affinity determination of small ligands in proteins21. Indeed, Trp has been primarily used as a fluorescence donor in FRET studies in proteins22,23,24, e. g., in terbium (Tb3+) FRET studies, a Trp residue is used frequently as an antenna for energy transfer to Tb3+ 25,26,27. Trp displays various advantages over other FRET donors due to its inherent constitutive character in the protein structure, which eliminates the need for preparative processes that may affect the function/structure of the studied protein24. Thus, the identification of radiative decays (energy transfer and changes in the medium polarity that are induced by protein structural rearrangements) is important for drawing accurate conclusions regarding ligand binding in protein structural studies13,14,19,28.
In protein structural studies, an extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), has been primarily used in experiments related to protein folding/unfolding28,29. ANS binds to proteins/enzymes in the native state, usually in the binding sites of substrates31,32,33; an increase in ANS fluorescence quantum yield (&#934;F) (namely, an increase in fluorescence intensity) is induced by exciting the protein at &#955;=370 nm when suitable interactions of ANS with Arg and His residues in hydrophobic pockets occur34,35,36,37. In various studies, the occurrence of FRET (when exciting at &#955; within 280-295 nm) between Trp residues (donors) and ANS (acceptor) has been reported, which is based on the following: 1) overlap of the fluorescence emission spectrum of Trp and excitation spectrum of ANS, 2) identification of a suitable distance between one or more Trp residue(s) and ANS for energy transfer, 3) high ANS quantum yield when bound in protein pockets, and 4) characteristic FRET pattern in the fluorescence spectra of the protein in the presence of ANS3,17,27,37,38.
Recently, ligand binding to the nucleotide-binding domain (N-domain) in SERCA and other P-type ATPases have been investigated using engineered recombinant N-domains40,41,42,43,44,45,46. Molecular engineering of the SERCA N-domain has been used to move the sole Trp residue (Trp552Leu) to a more dynamic structure (Tyr587Trp) that is close to the nucleotide-binding site, where fluorescence variations (quenching) may be used to monitor structural changes upon ligand binding34. Experimental results have demonstrated that ANS binds (as ATP) to the nucleotide-binding site in the purified recombinant SERCA N-domain34. Interestingly, the ANS fluorescence increases upon binding to the N-domain upon excitation at a &#955; of 295 nm, while the intrinsic fluorescence of the N-domain decreases34, thereby producing a FRET pattern that suggests the formation of a Trp-ANS FRET pair.
The use of NBS has been proposed to determine the content of Trp residues in proteins47 by absorbance assay of modified proteins. NBS modifies the highly absorbing indole group of Trp to the less absorbent oxindole47,48. This results in the loss (quenching) of the Trp fluorescent property40. Hence, NBS-mediated chemical modification of Trp residues may be used as an assay to define the role of Trp (as a donor) when FRET is hypothesized.
This protocol describes the chemical modification of the sole Trp residue by NBS in the engineered recombinant N-domain of SERCA as a protein model. Experimental results demonstrate that the ANS fluorescence intensity still increases in the chemically NBS-modified N-domain34, which lacks intrinsic fluorescence. Therefore, the assay is useful for demonstrating the absence of FRET between the Trp residue and ANS when bound to the N-domain34,40,49. Hence, this assay (NBS chemical modification of Trp) is useful in proving the presence of the Trp-ANS FRET pair in proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylamide</td>
			<td>Bio-Rad</td>
			<td>1610107</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>1610700</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>8-Anilino-1-naphthalenesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A1028</td>
			<td>Fluorophore</td>
		</tr>
		<tr>
			<td>Bis-acrylamide</td>
			<td>Bio-Rad</td>
			<td>1610125</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>N-Bromosuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>B81255</td>
			<td>Chemical modification</td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>J.T. Baker</td>
			<td>9213-12</td>
			<td>Stock solution preparation</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Chemical fluorescence label</td>
		</tr>
		<tr>
			<td>Fluorescence cuvette</td>
			<td>Hellma</td>
			<td>Z801291</td>
			<td>Fluorescence assay</td>
		</tr>
		<tr>
			<td>Fluorescence Spectrofluorometer</td>
			<td>Shimadzu</td>
			<td>RF 5301PC</td>
			<td>Fluorescence assay</td>
		</tr>
		<tr>
			<td>HisTrap&#8482; FF</td>
			<td>GE Healtcare</td>
			<td>11-0004-59</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>IPTG, Dioxane free</td>
			<td>American Bionalytical</td>
			<td>AB00841-00010</td>
			<td>Protein expression</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>LB media</td>
			<td>Fisher Scientific</td>
			<td>10000713</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pipetman L P10L</td>
			<td>Gilson</td>
			<td>FA10002M</td>
			<td>Fluorescence assay</td>
		</tr>
		<tr>
			<td>Pipetman L P100L</td>
			<td>Gilson</td>
			<td>FA10004M</td>
			<td>Fluorescence assay</td>
		</tr>
		<tr>
			<td>Pipetman L P200L</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td>Fluorescence assay</td>
		</tr>
		<tr>
			<td>Pipetman L P1000L</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td>Fluorescence assay</td>
		</tr>
		<tr>
			<td>Pipetman L P5000L</td>
			<td>Gilson</td>
			<td>FA10007</td>
			<td>Fluorescence assay</td>
		</tr>
		<tr>
			<td>Precision plus std</td>
			<td>Bio-Rad</td>
			<td>1610374</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate</td>
			<td>Bio-Rad</td>
			<td>1610302</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>J.T. Baker</td>
			<td>3828-19</td>
			<td>Buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>J.T. Baker</td>
			<td>3818-01</td>
			<td>Buffer preparation</td>
		</tr>
		<tr>
			<td>Syringe filter 0.2 um</td>
			<td>Millipore</td>
			<td>GVWP04700</td>
			<td>Solution filtration</td>
		</tr>
		<tr>
			<td>Temed</td>
			<td>Bio-Rad</td>
			<td>1610801</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Bio-Rad</td>
			<td>1610719</td>
			<td>SDS-PAGE</td>
		</tr>
	</tbody>
</table>

chemical modification of the tryptophan residue in a recombinant ca2+-atpase n-domain for studying tryptophan-ans fret

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional information may nonetheless be obtained from isolated recombinant domains. The engineered (Trp552Leu and Tyr587Trp) recombinant nucleotide-binding domain (N-domain) displays fluorescence quenching upon ligand binding. An extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), binds to the nucleotide-binding site via electrostatic and hydrophobic interactions with Arg, His, Ala, Leu, and Phe residues. ANS binding is evidenced by the increase in fluorescence intensity when excited at a wavelength (&#955;) of 370 nm. However, when excited at &#955; of 295 nm, the increase in fluorescence intensity seems to be coupled to the quenching of the N-domain intrinsic fluorescence. Fluorescence spectra display a F&#246;ster resonance energy transfer (FRET)-like pattern, thereby suggesting the presence of a Trp-ANS FRET pair, which appears to be supported by the short distance (~20 &#197;) between Tyr587Trp and ANS. This study describes an analysis of the Trp-ANS FRET pair by Trp chemical modification (and fluorescence quenching) that is mediated by N-bromosuccinimide (NBS). In the chemically modified N-domain, ANS fluorescence increased when excited at a &#955; of 295 nm, similar to when excited at a &#955; of 370 nm. Hence, the NBS-mediated chemical modification of the Trp residue can be used to probe the absence of FRET between Trp and ANS. In the absence of Trp fluorescence, one should not observe an increase in ANS fluorescence. The chemical modification of Trp residues in proteins by NBS may be useful for examining FRET between Trp residues that are close to the bound ANS. This assay will likely also be useful when using other fluorophores.

Molecular docking shows the binding of ANS to the nucleotide-binding site of the N-domain via electrostatic as well as hydrophobic interactions (Figure 1). Molecular distance (20 &#197;) between the Trp residue and ANS (bound to the nucleotide-binding site) supports the occurrence of FRET (Figure 1). The designed (engineered) recombinant N-domain was obtained at high purity by affinity chromatography (Figure 2) and was suitable for ...

Watch this Scientific Journal Video about Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET at JoVE.com

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

ANS binds to the Ca2+-ATPase recombinant N-domain. Fluorescence spectra display a FRET-like pattern upon excitation at a wavelength of 295 nm. NBS-mediated chemical modification of Trp quenches the fluorescence of the N-domain, which leads to the absence of energy transfer (FRET) between the Trp residue and ANS.

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional ...

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3.3K Views.  Universidad Autónoma de San Luis Potosí. ANS binds to the Ca2+-ATPase recombinant N-domain. Fluorescence spectra display a FRET-like pattern upon excitation at a wavelength of 295 nm. NBS-mediated chemical modification of Trp quenches the fluorescence of the N-domain, which leads to the absence of energy transfer (FRET) between the Trp residue and ANS.

Video: Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

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 Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

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 ...

FRET Analysis of IDR Structural Sensitivity to Study Hyperosmotic Stress in Yeast

Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical properties of the cellular environment change, directly impacting the conformational ensemble of IDRs. IDRs are inherently sensitive to environmental perturbations. Studying how the physicochemical properties of the cell regulate the conformational ensemble of IDRs is essential for understanding the environmental control of their function. Here, we describe a step-by-step method for measuring the structural sensitivity of IDRs in living Saccharomyces cerevisiae cells in response to hyperosmotic stress conditions. We present the use of ensemble fluorescence resonance energy transfer (FRET) to estimate how the global dimensions of IDRs change during a progressive increase of hyperosmotic stress imposed on cells with any osmolyte. In addition, we provide a script for processing fluorescence measurements and comparing structural sensitivity for different IDRs. By following this method, researchers can obtain valuable insights into the conformational changes that IDRs undergo in the complex intracellular milieu upon changing environments.

Author Spotlight: Unlocking the World of Intrinsically Disordered Regions with Cellular Sensing and Responses

Intrinsically disordered regions (IDRs) are flexible protein domains that modify their conformation in response to environmental changes. Ensemble fluorescence resonance energy transfer (FRET) can estimate protein dimensions under different conditions. We present a FRET approach to assess IDR structural sensitivity in living Saccharomyces cerevisiae cells under hyperosmotic stress.

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical ...