This study was supported by the Korea Research Fellowship (KRF) program, postdoctoral fellow of the National Research Foundation of Korea (NRF), funded by the ministry of science &#38; ICT (NRF-2019H1D3A1A01102952). The authors are thankful to KIST intramural grant and Ministry of Oceans and Fisheries grant number 2MRB130 for providing financial assistance for this project.

1. Lab-scale malachite green assay
<ol>
	<li>Preparation of assay buffer
	<ol>
		<li>Prepare the assay buffer, as per the composition and preparation presented in Table 1.</li>
	</ol>
	</li>
	<li>Preparation of phosphate standards
	<ol>
		<li>Use 1 mM phosphate standard, provided in the malachite green assay phosphate assay kit (stored at 4 &#176;C).</li>
		<li>Pipette 40 &#181;L of 1 mM phosphate standard in 960 &#181;L of ultra-pure water to obtain 40 &#181;M phosphate solution (premix solution). Add the premix solution with ultra-pure water, as per manufacturer&#39;s instructions, to form a serial dilution of phosphate from 0 to 40 &#181;M.</li>
	</ol>
	</li>
	<li>Preparation of yeast Hsp90
	<ol>
		<li>Use yeast Hsp90 with a glycerol stock concentration of 0.66 mg/mL. Thaw on ice just before use.</li>
		<li>Use 8 &#181;L (5.98 &#181;g, 0.80 &#181;M) of yeast Hsp90 for measurable ATPase activity. Check that the optical density difference after the addition of malachite green reagent between the blank and positive control is at least 0.5 and less than 1.0. Here, the absorbance difference between the blank (without Hsp90) and positive control (with Hsp90) was found to be 0.510.</li>
	</ol>
	</li>
	<li>Preparation of ATP
	<ol>
		<li>Transfer 1 mg of ATP to a vial containing 453.39 &#181;L of ultra-pure water to obtain 4 mM of stock solution. Perform weight calculations based on anhydrous ATP (molecular weight [m.w.] = 551.14). Prepare ATP fresh, as it can spontaneously hydrolyze over time.</li>
		<li>Add 4 &#181;L of ATP stock solution to each well to give a final well concentration of 0.2 mM (final total assay well volume of 80 &#181;L). Add ATP as the last component to the reaction, using a multichannel pipette so that all the reactions start simultaneously.</li>
	</ol>
	</li>
	<li>Preparation of geldanamycin (GA)
	<ol>
		<li>Prepare a 10 mM stock of GA by dissolving 1 mg in 178.37 &#181;L of DMSO (m.w. = 560.64). Using the stock solution, prepare 4 mM, 0.8 mM, 0.16 mM, 0.032 mM, 0.0064 mM, and 0.00128 mM solution in DMSO by serial dilution.</li>
		<li>Add 2 &#181;L of these stock solutions to each well to obtain a final well concentration of 0.1 mM, 0.02 mM, 0.004 mM, 0.0008 mM, 0.00016 mM, and 0.000032 mM, respectively.</li>
		<li>Prepare the wells as described in Table 2 and Table 3, with the wells containing the blank (buffer + water + DMSO) and negative control (Hsp90 in buffer + water + DMSO). The wells containing GA (Hsp90 in buffer + water + GA) serve as the positive control.</li>
	</ol>
	</li>
	<li>Preparation of well plates and order of addition
	<ol>
		<li>Prepare 96-well plates with the layout presented in Table 2. For compounds that show absorbance at 620 nm, prepare a separate well for recording the absorbance at this wavelength. Do not prepare a separate well for GA, as GA does not show absorbance at 620 nm for the concentration used in the assay.</li>
		<li>Prepare each constituent with the total volume of each constituent, as presented in Table 3.</li>
		<li>Set up assay wells by adding ultra-pure water to each well. Following this, add the required amount of assay buffer and a compound solution (for example, GA solution).</li>
		<li>Then, add Hsp90 to the appropriate wells, and shake the plates for 2 min on a plate shaker at room temperature.</li>
		<li>Add 4 &#181;L of ATP solution using a multi-channel pipette. Wrap the plate in aluminum foil and shake for 2 min on a plate shaker (200 rpm) at room temperature. Incubate the plate at 37 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Preparation and addition of malachite green reagent
	<ol>
		<li>The malachite green reagent consists of reagents A (ammonium molybdate in 3M HCl) and B (malachite green and polyvinyl alcohol). Bring the reagent to room temperature before use. Mix reagents A and B at a 100:1 ratio (1,000 &#181;L:10 &#181;L). Prepare this within 3 h before use, since it is unstable and starts decomposing after 3 h.</li>
		<li>After 3 h of step 1.6.5, stop the reaction by adding 20 &#181;L of malachite green reagent, added in the same order as ATP, using a multi-channel pipette.</li>
		<li>After a 15 min incubation at room temperature, measure the absorbance of the plate at 620 nm in a plate reader.</li>
	</ol>
	</li>
</ol>
2. High-throughput screening of Hsp90 inhibitors by malachite green assay
NOTE: The protocol for high-throughput screening is similar to the lab-scale methodology. The final well volume in each case is 80 &#181;L. However, there is a slight difference in the order of the addition of reagents. In the lab-scale based method, there are five stages of solution addition (34 &#181;L of water, 32 &#181;L of buffer, 2 &#181;L compound in DMSO, 8 &#181;L of Hsp90, and finally 4 &#181;L of ATP solution). In contrast, with HTS there are three stages of addition (40 &#181;L of buffer solution containing yeast Hsp90, 2 &#181;L of DMSO in 18 &#181;L of water containing the compounds, and finally 20 &#181;L of ATP dissolved in water). The minimum amount of solution that can be pipetted accurately in the HTS setup is 20 &#181;L. Hence, a difference in pipetting between lab and HTS scales is observed.
<ol>
	<li>Preparation of assay buffer (pH 7.4)
	<ol>
		<li>Use the same buffer in HTS as for the lab-scale malachite green assay (Table 1).</li>
	</ol>
	</li>
	<li>Preparation of yeast Hsp90
	<ol>
		<li>Use protein with a glycerol stock concentration of 0.66 mg/mL. Thaw on ice just before use.</li>
		<li>Use 8 &#181;L (5.98 &#181;g, 0.80 &#181;M) of yeast Hsp90 in each well, which provides measurable ATPase activity.</li>
		<li>Dilute the protein (8 &#181;L) in buffer (32 &#181;L) for each well. The final total working volume for each well is 40 &#181;L in a total assay volume of 80 &#181;L.</li>
	</ol>
	</li>
	<li>Preparation of 0.8 mM ATP
	<ol>
		<li>Dissolve 1 mg of ATP in 2,267 &#181;L of distilled water to obtain a 0.8 mM solution. The total working volume for each well is 20 &#181;L in a total assay volume of 80 &#181;L.</li>
	</ol>
	</li>
	<li>Preparation of geldanamycin (GA)/test compounds
	<ol>
		<li>Prepare a 0.8 mM stock of GA in DMSO, as in step 1.5. Dilute 10 &#181;L of the GA stock with 90 &#181;L of water to give a final concentration of 80 &#181;M.</li>
		<li>Use 20 &#181;L of the 80 &#181;M GA solution in appropriate assay wells (final total assay well volume = 80 &#181;L), giving a final well concentration of 20 &#181;M. This serves as a positive control.</li>
		<li>For test compounds, prepare solutions in DMSO as per their desired concentration for evaluation.</li>
		<li>Prepare 10 &#181;L of DMSO in 90 &#181;L of water for addition to blank (buffer + water + DMSO) and negative control wells (Hsp90 in buffer + water + DMSO). Prepare the test compounds in a similar fashion at a 100 &#181;M final well concentration.</li>
	</ol>
	</li>
	<li>Design of assay plates and order of addition
	<ol>
		<li>Use four main plates, one compound plate, and four addition plates containing stocks of reagents to set up the assay (Figure 2 and Table 4).</li>
		<li>In addition, plate A, add 90 &#181;L of assay buffer in each well; in addition plate B, add 90 &#181;L of buffer with Hsp90 in each well; in addition plates C and D, add 90 &#181;L of ATP and 90 &#181;L of malachite green reagent, respectively, in each well (Table 5 and Table 6).</li>
		<li>From addition plate A and plate B, transfer 40 &#181;L of solution from each well to main plate 1 and 2, and 3 and 4, respectively, using an automated multichannel dispensing system. Here, the Biomek(R) FXP laboratory automation workstation was used (Figure 2 and Table 6).</li>
		<li>From each well of the compound plate, transfer 20 &#181;L of the solution to main plates 1, 2, 3, and 4 (Figure 2). Shake the plates for 1 min in a shaker (200 rpm).</li>
		<li>From each well of the addition plate C (ATP), transfer 20 &#181;L of solution to main plates 1, 2, 3, and 4 (Figure 2). Shake the plates for 1 min in a shaker (200 rpm).</li>
		<li>Incubate the plates for 3 h at 37 &#176;C.</li>
		<li>Prepare the malachite green reagent as per step 1.7. After 3 h, transfer 20 &#181;L of malachite green reagent from addition plate D into the wells of main plates 1 ,2, 3 and 4 (Figure 2).</li>
		<li>After a 15 min incubation at room temperature, measure the absorbance of the plate is at 620 nm in a plate reader.</li>
	</ol>
	</li>
</ol>

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There are no competing financial interests.

Hsp90 is a significant target for the discovery of novel anticancer drug molecules. Since its druggability was established in 199410, 18 molecules have reached clinical trials. At present, seven molecules are in various phases of clinical trials, either alone or in combination22. All such small molecules are N-terminal ATP binding inhibitors. The other means of inhibiting the chaperone (C-terminal inhibitors, middle domain inhibitors) have not proceeded as fast as N-termina...

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains the stability of proteins responsible for the development and progression of cancer. In addition, proteins responsible for the development of resistance to antineoplastic agents are also clients of Hsp901. Hsp90 is overexpressed ubiquitously in all cancer cell types (&#62;90% of cellular proteins), compared to normal cells where it may constitute less than 2% of total proteins. Moreover, the Hsp90 of cancer cells resides in a complex with co-chaperones, whereas in a normal cell it is present predominantly in a free, un-complexed state2,3. In recent years, several Hsp90 inhibitors have been demonstrated to possess senolytic effects in in vitro and in vivo studies, where they have significantly improved the life span of mice4,5,6. All the aforementioned findings substantiate the fact that Hsp90 inhibitors could be effective in multiple cancer types, with fewer adverse effects and reduced chances of developing resistance. The chaperoning function of Hsp90 is accomplished by binding ATP at the N-terminal domain of Hsp90 and hydrolyzing it into ADP and free phosphate7. Small molecules that competitively bind to the ATP binding pocket of Hsp90 were found to successfully suppress the chaperoning effect of the protein. To date, this remains the best strategy for Hsp90 inhibition, which is supported by the fact that such inhibitors have reached clinical trials8. One of them, Pimitespib, was approved in Japan for the treatment of gastrointestinal stromal tumor (GIST) in June 20229. This is the first Hsp90 inhibitor approved since the druggability of the chaperone was established in 199410.
The malachite green assay is a simple, sensitive, fast, and inexpensive procedure for the detection of inorganic phosphate, suitable for automation and high-throughput screening (HTS) of compounds against its desired target11. The assay has been successfully employed for the screening of Hsp90 inhibitors in small lab-scale setups, as well as in a HTS12,13,14,15,16,17. The assay uses a colorimetric method that determines the free inorganic phosphate formed due to the ATPase activity of Hsp90. The basis of this quantification is the formation of a phosphomolybdate complex between free phosphate and molybdenum, which subsequently reacts with malachite green to generate a green color (Figure 1). This rapid color formation is measured on a spectrophotometer, or on a plate reader, between 600-660 nm18,19.
In the present protocol, the procedure for carrying out a malachite green assay with yeast Hsp90 and subsequent identification of inhibitors against the chaperone is described. The natural product molecule, geldanamycin (GA), with which the druggability of Hsp90 was first established, was taken as an authentic inhibitor10. HTS has become an integral part of the current drug discovery program, owing to the availability of a large number of molecules for testing. This technique has gained more significance in the past 2 years because of the urgent need for repurposing drugs for treating Covid-19 infection20,21. Therefore, a detailed outline for the HTS of molecules against yeast Hsp90 protein by adopting the malachite green assay method is presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1M Magnesium chloride solution in water</td>
			<td>Sigma-Aldrich</td>
			<td>63069-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Potassium chloride solution in water</td>
			<td>Sigma-Aldrich</td>
			<td>60142-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>SPL Life Sciences</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A7699-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biomek FX laboratory automation workstation</td>
			<td>Beckman Coulter</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Compounds 3-96</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>Histidine tagged yeast Hsp90 was obtained from Dr. Chrisostomos Prodromou, School of Life Sciences, University of Sussex, United Kingdom, and protein was expressed in KIST Gangneung Institute of Natural Products. Details cannot be disclosed due to patent infringement issues.</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Geldanamycin, 99% (HPLC), powder</td>
			<td>AK Scientific, Inc.</td>
			<td>V2064</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitroge UltraPure DNase/RNase-Free Distilled Water</td>
			<td>ThermoFisher Scientific</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Malachite Green Phosphate Assay&#160; Assay kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK307-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Detection Microplate Reader Synergy HT</td>
			<td>Biotek Instruments, Inc.</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HT multi-plate reader</td>
			<td>Biotek Instruments, Inc.</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochloride buffer solution, pH7.4</td>
			<td>Sigma-Aldrich</td>
			<td>93313-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Hsp90</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>School of Life Sciences, University of Sussex, United Kingdom and protein was expressed in KIST Gangneung Institute of Natural Products. Primary Accession number: P02829</td>
		</tr>
	</tbody>
</table>

malachite green assay for the discovery of heat-shock protein 90 inhibitors

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is dependent on its ability to hydrolyze adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and free phosphate. The ATPase activity of Hsp90 is linked to its chaperoning function; ATP binds to the N-terminal domain of the Hsp90, and disrupting its binding was found to be the most successful strategy in suppressing Hsp90 function. The ATPase activity can be measured by a colorimetric malachite green assay, which determines the amount of free phosphate formed by ATP hydrolysis. Here, a procedure for determining the ATPase activity of yeast Hsp90 by using the malachite green phosphate assay kit is described. Further, detailed instructions for the discovery of Hsp90 inhibitors by taking geldanamycin as an authentic inhibitor is provided. Finally, the application of this assay protocol through the high-throughput screening (HTS) of inhibitor molecules against yeast Hsp90 is discussed.

The results of the assay are interpreted in terms of absorbance due to free phosphate ion concentration. The absorbance by free phosphate due to ATP hydrolysis by the yeast Hsp90 at 620 nm is considered as 100% ATPase activity, or zero percentage protein inhibition. The inhibition of protein leads to the cessation of ATP hydrolysis (less free phosphate). which is reflected in terms of decreased absorbance at 620 nm.
Results of lab-scale malachite green assay 
The standard...

Watch this Scientific Journal Video about Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors at JoVE.com

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is ...

malachite-green-assay-for-discovery-heat-shock-protein-90

Research

JoVE Journal

Biochemistry

4.7K Views.  Korea Institute of Science and Technology (KIST). The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Video: Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

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

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

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

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

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 ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

Expanding the Mass Photometry Analysis Concentration Range with Microfluidics

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

Author Spotlight: Characterization of Low-Affinity Protein Interactions in Solution Using MassFluidix Technology

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

Assessment of Hsp70 Chaperone Activity Using Escherichia coli dnaK756 Cells

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

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Subtitles

Methods Article

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors