This study was supported by grants from Swedish Research Council (2018-02843), Brain Foundation (Fo 2019-0140), Foundation for Geriatric Diseases at Karolinska Institutet, Gunvor and Josef An&#233;rs Foundation, Magnus Bergvalls Foundation, Gun and Bertil Stohnes Foundation, Tore Nilssons Foundation for medical research, Margaretha af Ugglas foundation and the Foundation for Old Servants.

NOTE: An overview of this protocol is shown in Figure 2.
1. Expression and purification of GST-FKBP51 and GST-FKBP52 (Figure 2A)
<ol>
	<li>Plasmids 
	NOTE: Obtain cDNA clones for human FKBP51 (clone id: 5723416) and for human FKBP52 (clone id: 7474554) from IMAGE consortium.
	<ol>
		<li>Amplify the human FKBP51 DNA by PCR with primers (forward; 5`GGATCCATGACTACTGATGAAGGT-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing BamHI and XhoI overhangs and clone into pGEX6-1 vector at BamHI / XhoI restriction sites.</li>
		<li>Amplify the human FKBP52 DNA by PCR with primers (forward; 5`-GAATTCATGACAGCCGAGGAGATG-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing EcoRI and XhoI overhangs and clone into pGEX6-2 vector at EcoRI / XhoI restriction sites. 
		NOTE: PCR reaction set up and conditions are shown in Table 1 and Table 2.</li>
		<li>Verify the inserted sequence and transform the plasmids into the chemically competent E. coli&#160;according to the manufacture protocol.</li>
	</ol>
	</li>
	<li>Protein expression and purification
	<ol>
		<li>Add 25 g of Luria broth (LB) base in 1 L of distilled water to make the LB solution. Autoclave it at 121 &#176;C for 15 min. After cooling, add 50 &#181;g/mL ampicillin.</li>
		<li>Take a colony of bacteria expressing GST-FKBP51 or GST-FKBP52 and mix with 500 &#181;L of LB solution in a 1.5 mL tube. Vortex.</li>
		<li>Add the mixture of &#34;1.2.2&#34; into 1 L of LB solution in the Erlenmeyer flask covered with an aluminum foil. Incubate the Erlenmeyer flask in the shaker overnight at 37 &#176;C.</li>
		<li>Induce protein expression by adding 1 mM isopropyl-&#946;-D-thiogalactoside (IPTG) to the Erlenmeyer flask and continue the incubation for a further 2 h.</li>
		<li>To get cell pellets, centrifuge at 5,000 x g for 15 min. Remove the supernatant. 
		NOTE: The cell pellets can be stored at -20 &#176;C.</li>
		<li>Resuspend the cell pellets in 40 mL of PBS and sonicate 3 x 20 s on ice. Add 1 mM PMSF, 1 mM EDTA, and protease inhibitor cocktail (1 tablet) to prevent proteolysis.</li>
		<li>Centrifuge the suspension for 30 min 50,000 x g to remove cell debris and apply the supernatant onto 5 mL GST-trap column.</li>
		<li>After washing the column with 30 mL PBS, elute GST-FKBP51 and GST-FKBP52 with 5 mL of 10 mM glutathione in PBS.</li>
		<li>Concentrate proteins on 15 mL 10.000 MWCO centrifugation unit. To remove free glutathione, pass concentrates through PD-10 column equilibrated with 0.5x PBS and again concentrate on the filter centrifuge device.</li>
		<li>Collect protein-containing fractions. Verify the proteins in SDS-PAGE and adjust protein concentrations to 1 mg/mL. 
		&#8203;NOTE: Typical protein yield is 2-5 mg/L culture. The protein can be stored at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Coupling of Hsp90 C-terminal peptide to the acceptor beads (Figure 2B)
<ol>
	<li>Hsp90 peptide preparation
	<ol>
		<li>Synthesize ten amino acid peptide NH2-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform (UniProt ID: P08238) by a peptide synthesis service.</li>
		<li>Dilute the Hsp90 peptide in PBS to 1 mg/mL concentration.</li>
	</ol>
	</li>
	<li>Acceptor beads preparation
	<ol>
		<li>Dilute the unconjugated acceptor beads in PBS to 1 mg/mL concentration and transfer to a 1.5 mL tube.</li>
		<li>Perform the washing by centrifugation at 16,000 x g for 15 min. Carefully remove the supernatant.</li>
	</ol>
	</li>
	<li>Conjugation
	<ol>
		<li>Set the ratio between beads and peptide as 10:1. In the 1.5 mL tube containing 1 mg of acceptor bead pellet (prepared as described above), add 1 mL of PBS (pH 7.4), 0.1 mg of diluted peptide, 1.25 &#181;L of Tween-20, 10 &#181;L of a 400 mM solution of sodium cyanoborohydride (NaBH3CN) in water. 
		CAUTION: NaBH3CN is toxic; use a fume hood and gloves. NaBH3CN solution should be freshly prepared.</li>
		<li>Incubate for 24 h at 37 &#176;C with end-over-end agitation (10-20 rpm) on a rotary shaker.</li>
	</ol>
	</li>
	<li>Reaction quenching and beads washing
	<ol>
		<li>Add 20 &#181;L of 1 M Tris-HCl (pH 8.0) solution to the reaction to block unreacted sites. Incubate for 1 h at 37 &#176;C.</li>
		<li>Centrifuge at 16,000 x g (or maximum speed) for 15 min at 4 &#176;C. Remove the supernatant and resuspend the bead pellet in 1 mL of Tris-HCl solution (100 mM, pH 8.0).</li>
		<li>Repeat the washing step three times.</li>
		<li>After the last centrifugation, resuspend the beads at 1 mg/mL in storage buffer (1 mL of 0.5 &#215; PBS with 0.01% sodium azide as a preservative). Store the conjugated acceptor bead solution at 4 &#176;C light protected. 
		&#8203;CAUTION: Sodium azide is toxic; use a fume hood and gloves.</li>
	</ol>
	</li>
</ol>
3. The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide, and inhibition with small molecular mass compounds (Figure 2C)
<ol>
	<li>GST-tagged proteins interacting with glutathione donor beads
	<ol>
		<li>Set up the reactions in 384-well plates.</li>
		<li>Prepare the solution containing 10 &#181;g/mL of the glutathione donor beads in 0.5x PBS, pH 7.4. 
		NOTE: After prolonged storage, beads settle down and need to be vortexed.</li>
		<li>Add GST-FKBP51 or GST-FKBP52 to a final concentration of 10 &#181;g/mL.</li>
		<li>Incubate in the dark at 25 &#176;C for 10 min. 
		NOTE: At this step, GST-tagged proteins will interact with glutathione attached to the beads. For each well, 22.5 &#181;L of this mixture will be used. The concentration of the binding partners must be determined empirically. Titrate GST-FKBP51 and GST-FKBP52 and choose the concentration that gives the best signal.</li>
	</ol>
	</li>
	<li>Compound addition
	<ol>
		<li>Make serial dilutions of test compounds in DMSO. 
		NOTE: The concentrations used are typically 10, 30, 100, 300, 1,000, and 3,000 &#181;M.</li>
		<li>Add 0.25 &#181;L of DMSO (negative control) or Hsp90 C-terminal peptide (positive control, 30 &#181;M) or compounds in DMSO to the corner of each well of the plate. Use triplicates for every compound concentration.</li>
		<li>Add 22.5 &#181;L of the solution containing glutathione donor beads with GST-tagged proteins to each well.</li>
		<li>Shake the plate gently with hand but thoroughly. Incubate in the dark at 25 &#176;C for 15 min. 
		&#8203;NOTE: During this time, compounds will interact with the TPR domain at the Hsp90 C-terminal peptide binding site.</li>
	</ol>
	</li>
	<li>Acceptor beads addition
	<ol>
		<li>Dilute the acceptor beads with attached Hsp90 C-terminal peptide to 100 &#181;g/mL in 0.5x PBS.</li>
		<li>Add 2.25 &#181;L of diluted acceptor beads to each well.</li>
		<li>Mix gently but thoroughly. Incubate in the dark at 25 &#176;C for 15 min. 
		NOTE: At this step, donor and acceptor beads are brought into proximity by the protein-peptide interactions. The final volume of the reaction mixture is 25 &#181;L. Therefore, the final concentrations of compounds are ranging from 0.1 to 30 &#181;M.</li>
	</ol>
	</li>
	<li>Plate reading 
	NOTE: Read the plate using a plate reader set in the relevant mode.
	<ol>
		<li>Turn on the instrument and open the software</li>
		<li>Choose the relevant protocol.</li>
		<li>Click Edit plate map and select the well being used in the plate for measurement.</li>
		<li>Click Next to continue and Run the selected protocol.</li>
		<li>After measurement, click Show Results to view results.</li>
		<li>Export the data.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Z&#39; factor and signal-to-background (S/B) ratio
	<ol>
		<li>Calculate the Z&#39; factor and S/B ratio for the assay using the following equation: 
		Z&#39;=1-(3&#963;pos+3&#963;neg)/&#9474;&#181;pos-&#181;neg&#9474;16 
		S/B=&#181;neg/&#181;pos 
		where, &#963; and &#181; represent the standard deviations and means of the positive (Hsp90 C-terminal peptide, 30 &#181;M) and negative (DMSO) controls, respectively. A Z&#39; factor &#62; 0.5 will ensure that the assay is robust enough for screening. To monitor the assay sensitivity, the S/B ratio has also been calculated.</li>
	</ol>
	</li>
	<li>Dose-response curve and IC 50 
	NOTE: Use nonlinear regression analysis to fit the data of Hsp90-cochaperones PPI inhibitors by software.
	<ol>
		<li>Create an XY data table in the Welcome dialog and select X Numbers, and Y Enter 3 (if triplicates) replicate values in side-by-side columns.</li>
		<li>Normalize the signal data of samples to the negative control group. Import concentration values to the X column and the signal values to the Y column.</li>
		<li>Click Analyze and choose Transform concentration (X) under Transform | Normalize. Choose Transform to Logarithms. 
		NOTE: This will transform the concentration to a log scale. If your starting concentration is zero, set it to a very small number that is effectively zero (e.g., 0.1 nM) not to lose those values since the logarithm of zero is not defined.</li>
		<li>Click Analyze and choose Nonlinear regression (curve fit) under XY analyses, open the Dose-Response-Inhibition and choose Log(inhibitor) vs. response -- Variable slope.</li>
		<li>Click OK to view the Results (containing IC50 value) and Graphs.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62762/62762fig2v2.jpg" /> 
Figure 2: Schematic of this protocol. (A) Expression and purification of GST-FKBP51 and GST-FKBP52. (B) Coupling of Hsp90 C-terminal peptide to the acceptor beads. (C) The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide. Inhibition with small molecular mass compounds. Created with BioRender.com <a href="https://www.jove.com/files/ftp_upload/62762/62762fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Authors report no conflicts of interest.

Here we describe a protocol using the assay for screening small molecules inhibiting interactions between Hsp90 and TPR-motif co-chaperones, especially FKBP51 and FKBP52. Its high Z' score (&gt;0.8) demonstrates the robustness and reliability for a high-throughput format. Results can be obtained within one hour, and small amounts of beads, protein and compounds are required. Moreover, this protocol could easily be extended to any Hsp90/Hsp70 - TPR-motif co-chaperone interactions of interest. Several TPR-motif co-chaperon...

Molecular chaperones contribute to protein homeostasis, including protein folding, transport, and degradation. They regulate several cellular processes and are linked to numerous diseases such as cancer and neurodegenerative diseases1. Heat shock protein 90 (Hsp90) is one of the most important chaperones whose function is dependent on conformational changes driven by ATP hydrolysis and binding with client proteins mediated by its co-chaperones2. Despite an obvious potential of Hsp90 as the therapeutic target, fine-tuning its function represents a big challenge. There are several Hsp90 inhibitors targeting the N-terminal ATP binding region, which have been evaluated in clinical trials, but none of them has been approved for marketing3. Due to the lack of a well-defined ligand-binding pocket4, targeting the C-terminal region of Hsp90 has had limited success4. Recently, disruption of Hsp90-cochaperone interactions by small molecules has been investigated as an alternative strategy5. Targeting the Hsp90-cochaperone interactions would not elicit general cell stress response and provides the possibility to specifically regulate various intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones6. Of the 736 TPR motif-containing proteins annotated in the human protein database, ~20 different proteins interact with Hsp90 via this peptide7. Molecules competing for MEEVD peptide-binding would disrupt the interactions between Hsp90 and co-chaperones containing a TPR domain. The peptide binding site has similar tertiary structure but the overall homology between different TPR motif domains is relatively low7, providing an opportunity to identify molecules specifically capable of blocking interactions between Hsp90 and particular TPR-motif co-chaperones. Among these TPR-motif co-chaperones, FK506-binding protein (FKBP) 51 and FKBP52 are regulators of steroid hormone receptor (SHR) signaling and involved in several steroid hormone-dependent diseases including cancer, stress-related diseases, metabolic diseases, and Alzheimer&#39;s disease8. Although FKBP51 and FKBP52 share &#62; 80% sequence similarity, their functions differ: FKBP52 is a positive regulator of SHR activity, while FKBP51 is a negative regulator in most cases8. Therefore, identifying molecules, specifically blocking interactions between Hsp90 and FKBP51 or FKBP52, provides a promising therapeutic potential for related diseases.
Amplified Luminescent Proximity Homogenous Assay (AlphaScreen) was first developed in 1994 by Ullman EF et al.9. Now it is widely used to detect different types of biological interactions, such as peptide10, protein11, DNA12, RNA13, and sugar14. In this technique, there are two kinds of beads (diameter 200 nm), one is the donor bead and the other is the acceptor bead. The biomolecules are immobilized onto these beads; their biological interactions bring donor and acceptor beads into proximity. At 680 nm, a photosensitizer in the donor bead illuminates and converts oxygen to singlet oxygen. Because the singlet oxygen has a short lifetime, it can only diffuse up to 200 nm. If the acceptor bead is in proximity, its thioxene derivative reacts with the singlet oxygen generating chemiluminescence at 370 nm. This energy further activates fluorophores in the same acceptor bead to emit light at 520-620 nm15. If the biological interactions are disrupted, the acceptor bead and donor bead cannot reach proximity, resulting in the singlet oxygen decay and low produced signal.
Here we describe a protocol using this technique for screening small molecules inhibiting interactions between Hsp90 and TPR co-chaperones, especially FKBP51 and FKBP52.The 10 amino acid long peptides corresponding to Hsp90 extreme C-terminus are attached to acceptor beads. Purified GST-tagged TPR co-chaperones interact with glutathione-linked donor beads. When the interaction between Hsp90-derived peptides and TPR-motif co-chaperones brings the beads together, an amplified signal is produced (Figure 1A). If the screened small molecules can inhibit the interactions between Hsp90 and TPR-motif co-chaperones, this amplified signal will be decreased (Figure 1B). Their IC50 can be calculated by quantitative measurement. This protocol can be extended to any chaperone - TPR-motif co-chaperone interactions of interest and is of great importance in the development of novel molecules, specifically blocking the interaction between Hsp90 and FKBP51 or FKBP52.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62762/62762fig01.jpg" /> 
Figure 1: The basic principle of this assay. (A) Purified GST-FKBP51 interacts with glutathione-linked donor beads. The 10 amino acid long peptides corresponding to the extreme C-terminus of Hsp90 are attached to acceptor beads. The interaction between Hsp90-derived peptides and TPR domain of FKBP51 brings the donor and acceptor beads into proximity. At 680 nm, a photosensitizer in the donor bead illuminates and converts oxygen to singlet oxygen. The thioxene derivative on the acceptor bead reacts with the singlet oxygen and generates chemiluminescence at 370 nm. This energy further activates fluorophores in the same acceptor bead to emit light at 520-620 nm. (B) When small molecules inhibit the interactions between Hsp90 and FKBP51, the donor and acceptor beads cannot reach proximity. Then the singlet oxygen with short lifetime decays, and no detectable signal is produced. <a href="https://www.jove.com/files/ftp_upload/62762/62762fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>384-well plates</td>
			<td>Perkin Elmer</td>
			<td>6008350</td>
			<td>Assay volume 25 ml</td>
		</tr>
		<tr>
			<td>Amicon 10.000 MWCO centrifugation unit</td>
			<td>Millipore</td>
			<td>UFC901008</td>
			<td>Concentrate protein</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Bacteria shaker Unimax 1010</td>
			<td>Heidolph</td>
			<td></td>
			<td>Culture bacteria</td>
		</tr>
		<tr>
			<td>cDNA clones for human FKBP51</td>
			<td>Source BioScience</td>
			<td>clone id: 5723416</td>
			<td>pCMV-SPORT6 vector</td>
		</tr>
		<tr>
			<td>cDNA clones for human FKBP52</td>
			<td>Source BioScience</td>
			<td>clone id: 7474554</td>
			<td>pCMV-SPORT6 vector</td>
		</tr>
		<tr>
			<td>Chemically Competent E. coli</td>
			<td>Invitrogen</td>
			<td>C602003</td>
			<td>One Shot BL21 Star (DE3)</td>
		</tr>
		<tr>
			<td>Data analysis software</td>
			<td>GraphPad Prism</td>
			<td>9.0.0</td>
			<td>Analysis data and make figures</td>
		</tr>
		<tr>
			<td>Data analysis software</td>
			<td>Excel</td>
			<td></td>
			<td>Analysis data</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Supelco</td>
			<td>1.02952.1000</td>
			<td>Dilute compounds</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Prepare solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Calbiochem</td>
			<td>344504</td>
			<td>Prevent proteolysis during sonication</td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>Sigma</td>
			<td>G-4251</td>
			<td>Elute GST-tagged proteins</td>
		</tr>
		<tr>
			<td>Glutathione donor beads</td>
			<td>Perkin Elmer</td>
			<td>6765300</td>
			<td>Donor bead</td>
		</tr>
		<tr>
			<td>GST-trap column</td>
			<td>Cytiva (GE Healthcare)</td>
			<td>17528201</td>
			<td>Purify GST-tagged proteins</td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactoside</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0392</td>
			<td>Induce protein expression</td>
		</tr>
		<tr>
			<td>LB Broth (Miller)</td>
			<td>Sigma</td>
			<td>L3522</td>
			<td>Microbial growth medium</td>
		</tr>
		<tr>
			<td>PCR instrument</td>
			<td>BIO-RAD</td>
			<td>S1000 Thermal Cycler</td>
			<td>Amplification/PCR</td>
		</tr>
		<tr>
			<td>PD-10 column</td>
			<td>Cytiva (GE Healthcare)</td>
			<td>17085101</td>
			<td>Solution exchange</td>
		</tr>
		<tr>
			<td>pGEX-6P-1 vector</td>
			<td>Cytiva (GE Healthcare)</td>
			<td>28954648</td>
			<td>Plasmid</td>
		</tr>
		<tr>
			<td>pGEX-6P-2 vector</td>
			<td>Cytiva (GE Healthcare)</td>
			<td>28954650</td>
			<td>Plasmid</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Perkin Elmer</td>
			<td>EnSpire 2300 Multilabel Reader</td>
			<td>Read alpha plate</td>
		</tr>
		<tr>
			<td>Plate reader software</td>
			<td>Perkin Elmer</td>
			<td>EnSpire Manager</td>
			<td>Plate reader software</td>
		</tr>
		<tr>
			<td>Plate reader software protocol</td>
			<td>Perkin Elmer</td>
			<td>Alpha 384-well Low volume</td>
			<td>Use this protocol to read plate</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>Prevent proteolysis during sonication</td>
		</tr>
		<tr>
			<td>protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>S8830</td>
			<td>Prevent proteolysis during sonication</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>As a preservative</td>
		</tr>
		<tr>
			<td>Sodium cyanoborohydride (NaBH3CN)</td>
			<td>Sigma</td>
			<td>156159</td>
			<td>Activates matrix for coupling</td>
		</tr>
		<tr>
			<td>Ten amino acid peptide NH2-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform</td>
			<td>Peptide 2.0 inc</td>
			<td></td>
			<td>Synthesize Hsp90 C-terminal peptide</td>
		</tr>
		<tr>
			<td>Test-Tube Rotator</td>
			<td>LABINCO</td>
			<td></td>
			<td>Make end-over-end agitation</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>10708976001</td>
			<td>Block unreacted sites of acceptor beads</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td>Prevent beads aggregation</td>
		</tr>
		<tr>
			<td>Ultra centrifuge Avanti J-20 XP</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Centrifuge to get bacteria cell pellets</td>
		</tr>
		<tr>
			<td>Ultrasonic cell disruptor</td>
			<td>Microson</td>
			<td></td>
			<td>Sonicate cells to release protein</td>
		</tr>
		<tr>
			<td>Unconjugated acceptor beads</td>
			<td>Perkin Elmer</td>
			<td>6762003</td>
			<td>Acceptor beads</td>
		</tr>
		<tr>
			<td>XCell SureLock Mini-Cell and XCell II Blot Module</td>
			<td>Invitrogen</td>
			<td>EI0002</td>
			<td>SDS-PAGE</td>
		</tr>
	</tbody>
</table>

studies of chaperone-cochaperone interactions using homogenous bead-based assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

In our assay, Z&#39; factor and S/B ratio are 0.82 and 13.35, respectively (Figure 3A), demonstrating that our assay is robust and reliable for high-throughput screening. We then used it to screen small molecular mass compounds. Figure 3B presents dose-dependent inhibition of chaperone-cochaperone interactions with a selected small molecule (D10). The dose-response curves for D10 are generated by nonlinear regression analysis, based on which the values of IC<sub...

Watch this Scientific Journal Video about Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay at JoVE.com

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

studies-chaperone-cochaperone-interactions-using-homogenous-bead

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JoVE Journal

Biochemistry

2.6K Views.  Karolinska Institutet. This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Video: Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies.

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface.

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure ...