Name: studies of chaperone-cochaperone interactions using homogenous bead-based assay
Uploaded: 2021-07-21T13:00:00
Description: Watch this Scientific Journal Video about Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay at JoVE.com

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...

<ol>
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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+binding+of+tetratricopeptide+repeat+proteins+to+the+C-terminal+12-kDa+domain+of+hsp90.">Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90.</a> Journal of Biological Chemistry. 273 (29), 18007-18010 (1998).</li><li>Scheufler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+TPR+domain-peptide+complexes:+critical+elements+in+the+assembly+of+the+Hsp70-Hsp90+multichaperone+machine.">Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine.</a> Cell. 101 (2), 199-210 (2000).</li><li>Storer, C. L., Dickey, C. A., Galigniana, M. D., Rein, T., Cox, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FKBP51+and+FKBP52+in+signaling+and+disease.">FKBP51 and FKBP52 in signaling and disease.</a> Trends in Endocrinology &amp; Metabolism. 22 (12), 481-490 (2011).</li><li>Ullman, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescent+oxygen+channeling+immunoassay:+measurement+of+particle+binding+kinetics+by+chemiluminescence.">Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (12), 5426-5430 (1994).</li><li>Wigle, T. 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L., Shelat, A. A., Guy, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+Optimization+and+Screening+of+RNA-Protein+Interactions+by+AlphaScreen.">Assay Optimization and Screening of RNA-Protein Interactions by AlphaScreen.</a> Journal of Biomolecular Screening. 12 (7), 946-955 (2007).</li><li>Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+competitive+alphascreen+assay+for+detection+of+hyaluronan.">A competitive alphascreen assay for detection of hyaluronan.</a> Glycobiology. 28 (3), 137-147 (2018).</li><li>Principles of alphascreen amplified luinescent proximmity homogenous assay. 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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 ...

We built a homogeneous bead-based assay to study chaperone-cochaperone interactions. Using this technique, we screen small molecules inhibiting interactions between Hsp90 and FKDP51 or FKBP52 and identify potent and selective inhibitors. This high throughput screening technique is robust and reliable.Results can be obtained within one hour, and it only requires small amounts of beads protein and compounds. Hsp90 interaction with this cochaperones was implicated in human disorders, such as Alzheimer's disease, Cancer, Autoimmune disease. This technique provides the opportunity to screen chaperone-cochaperone inhibitors with high medical importance.Here is the basic principle of this assay. When the interaction between Hsp90C-Terminal peptide and TPR domain of cochaperones brings the donor and acceptor beads into proximity, a highly amplified signal is generated. When the interaction inhibitors are added, the donor and acceptor beads cannot reach proximity, and no detectable signal is produced.Begin by diluting the Hsp90 peptide in PBS at one milligram per milliliter concentration. Dilute the unconjugated acceptor beads in PBS, and transfer the beads into a 1.5 milliliter tube. Centrifuge the diluted beads for washing, and then carefully remove the supernatant.For conjugating, set the acceptor beads in peptides at a ratio of 10 to 1. To the paletide beads at PBS diluted peptide, Tween 20 and Sodium cyanaboroughhydride solution, and incubate with end over end agitation on a rotary shaker. To block the unreacted sites, add 20 microliters of one molar Tris hydrochloride solution of pH 8.0 to the beads, and quench the reaction by incubating for one hour at 37 degrees Celsius.At the end of the incubation, wash the beads by centrifugation and re-suspend the bead pellet in one milliliter of Tris hydrochloride solution. Repeat the washing two more times. After the last washing, re-suspend the beads at one milligram per milliliter concentration in storage buffer and store the conjugated acceptor bead solution at 4 degrees Celsius protected from light.Add glutathione donor beads at 10 micrograms per milliliter concentration in PBS. Add GSTFKBP51 to a final concentration of 10 micrograms per milliliter and incubate the reaction for 10 minutes at 25 degrees Celsius in the dark. Make serial dilutions of the test compound in DMSO.Add 0.25 microliters of test compound dilutions and triplicate in the corner of each well of a 384 well plate. For negative control, add 0.25 microliters of DMSO and for positive control, add 0.25 microliters of Hsp90 C-Terminal peptide in the wells. Add 22.5 microliters of the solution containing glutathione donor beads with GST tagged proteins to each well.Shake the plate thoroughly with hands and incubate in the dark at 25 degrees Celsius for 15 minutes. Dilute the acceptor beads with the attached Hsp90C-Terminal peptide to 100 micrograms per milliliter concentration in 0.5 times PBS. Add 2.25 microliters of diluted acceptor beads to each well.Shake and incubate the plate in the dark for 15 minutes as demonstrated. Turn on the plate reader instrument, measure the signal of the plate and analyze the data as described in the text manuscript. Create an X-Y data table in the welcome dialog, select X numbers, Y and enter three replicate values in side-by-side columns.Import the concentration values to the X column and the signal values to the Y column. Click analyze, select transform concentration X, and then select transform to logarithms. Click analyze and choose nonlinear regression under X-Y analysis.Open the dose response inhibition option and choose log versus response variable slope equation. Click okay to view the results containing IC50 value and graphs. This assay was used for screening small molecules, inhibiting interactions between Hsp90 and FKBP51 or FKBP52.The Z score of more than 0.8 and signal to background ratio of 13.35, demonstrate the robustness and reliability for high throughput screening. The effect of small molecular weight test compound D10 on inhibition of chaperone-cochaperone interactions was assessed and IC50 values were calculated, where D10 showed dose dependent inhibition. The IC50 value for Hsp90 GSTFKBP51 interactions was 65 nanomolar.Whereas for Hsp90, GSTFKBP52 interactions, complete inhibition was not achieved with the highest compound concentration indicates that selective inhibition of protein-protein interaction with small molecules can be achieved. The critical step is the order of addition. We first form a complex between a small molecule and its TPR target.Otherwise longer incubation and higher compound concentrations are required. We can use this technique to screen for small molecules disrupting the interaction between Hsp90 and FKB51 or Hsp90 and FKB52. It can also be extended to any interaction between Hsp90, Hsp70 and a TPR motif containing cochaperone screening selective inhibitors.

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

Research

JoVE Journal

Biochemistry

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 ...