We thank the National Institutes of Health (1R21CA138373 and Stand Up to Cancer (SU2C) for financial supports of this work. H.Y. is grateful for the 2009 Elion Award from the American Association of Cancer Research, a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research (SKF-08-101), and the National Science Foundation Faculty Early Career Award (NSF0954819).

1. Cloning Considerations
<ol>
 <li>Commercially prepared oligonucleotides representing the TMD of interest flanked by NheI and BamHI restriction sites and 5'-phosphorylated can be ligated into pTox7 (modified in our laboratory by insertion of one base pair directly after the BamHI restriction site14) (Figure 3) digested sequentially with BamHI and NheI. An example oligonucleotide is shown below: 
 5'ctagcTMDSEQUENCEg3' 
 3' gTMDSEQUENCEcctag5'</li>
</ol>
The TMD sequence should be 12-24 residues (shorter sequences will presumably be elongated by vector encoded hydrophobic residues). In order to investigate the interface, four variants of the TMD design should be created where sequential residue insertions and concomitant residue deletions result in rotation of the TMD relative to ToxR.15,16 Finally, the arabinose concentration should be varied between 0.001 and 0.01% (w/v) to identify the concentration where maximum differences in &#946;-galactosidase signals between different TMD sequences are observed; testing different expression levels is recommended to identify conditions under which different affinities can be distinguished best. In addition to arabinose and antibiotics, 0.4 mM IPTG can be used to enhance differences of affinities between different TMDs. The ToxR measurement should be performed at least in quadruplicate. The whole procedure should be repeated at least three times with different plasmid transformations.
2. Growth of Bacterial Cultures
<ol>
 <li>Gently thaw FHK12 competent cells (200 &mu;l) on ice and transfer into a 15 ml culture tube. Add plasmid DNA (200 ng) and incubate the cells on ice for 30 min.</li>
 <li>Heat-shock cells by incubation for 90 s at 42 &deg;C, followed by incubation on ice for 2 min.</li>
 <li>Add SOC media (800 &mu;l) and incubate the samples at 37 &deg;C with shaking (300 rpm) for one h.</li>
 <li>Inoculate 5 ml LB media with chloramphenicol (30 &mu;g/ml) and arabinose (0.0025% w/v) with 50 &mu;l of the transformation mixture in 15 ml culture tubes in triplicate. Incubate samples at 37 &deg;C with shaking (300 rpm) for 20 h. (Alternatively 5 &mu;l of culture can be used to inoculate 100 &mu;l of medium in a 96-well plate. This method is useful when dealing with large numbers of samples, although errors will be slightly higher. In order to avoid evaporation, which would lead to error, fill the outermost wells with media, but do not use them for samples. Finally, double-wrap the joint between the lid and the plate with parafilm).</li>
</ol>
3. Measurement of &#946;-galactosidase Activity
<ol>
 <li>Preheat the plate-reader to 28 &deg;C.</li>
 <li>Transfer the Z-buffer into a reservoir with a large pipette tip, making sure to only take up the upper (aqueous) layer. Transfer 100 &mu;l of freshly prepared Z-buffer/chloroform to the wells of a 96-well plate. Transfer 5 &mu;l of each culture into the wells of the plate in quadruplicate. Omit the culture from four wells which will serve as the blank.</li>
 <li>Measure the OD595 of the plate to determine cell density.</li>
 <li>Add 50 &mu;l of Z-buffer/SDS to all wells of the plate. Shake the plate in the plate-reader for 10 min to lyze the cells. Make sure the cell suspensions are clear after lysis and repeat the shaking step if required. Incomplete lysis suggests the Z-buffer/chloroform was not freshly prepared.</li>
 <li>Add 50 &mu;l freshly prepared Z-buffer/ONPG to all wells and return the plate to the plate reader and measure OD405 every 30 s for 20 min.</li>
 <li>Calculate &#946;-galactosidase activity using the following equation (remembering to subtract the blank). The ratio of OD405/min should be calculated using all data points in the OD405 range 0.0 to 1.0 using a linear model fit.
 
<img src="/files/ftp_upload/2721/2721eq1.jpg" alt="Equation 1" />
 
 Miller units differ sometimes when recorded on different days. Therefore, a reference construct like GpA should be measured in each test. Its values can be used for normalization of ToxR values.</li>
</ol>
4. Control for Protein Expression
<ol>
 <li>Perform Western blotting to verify even protein expression between constructs. Combine 50 &mu;l of the triplicate cultures and centrifuge (2000 rpm, 4 min) in a microcentrifuge. Remove the supernatant by pipetting and resuspend the residual pellet in 2 x sample loading buffer.</li>
 <li>Load 7.5 &mu;l on a standard 8% gel and carry out electrophoreses at 125 V for 1 hour 5 min. After transfer, incubate with anti-MBP HRP-conjugated antibody and visualize; the chimeric protein is observed at approximately 70 kDa with some degradation products sometimes seen around 48 kDa. Endogenous MBP is also observed at 45 kDa (see Figure 5).</li>
</ol>
5. Control for Proper Membrane Insertion
A cell line deficient in maltose binding protein is used to assess proper membrane insertion of the chimeric TMD construct. When grown on minimal media with maltose as the sole carbon source, only cells expressing a membrane-integral expression product with maltose binding protein correctly located to the periplasm are able to grow.
<ol>
 <li>Transform PD28 cells (as described for FHK12 cells) and inoculate 2 ml of LB medium. Grow the cells at 37 &deg;C with shaking (300 rpm) overnight.</li>
 <li>Pellet the cells by centrifugation at 3500 rpm, 10 min, 4 &deg;C and wash by resuspension in PBS (2 ml) by gentle pipetting with a large tip or gentle vortexing. Pellet the cells (as above), wash with PBS for a second time, pellet and finally resuspend in PBS (1 ml).</li>
 <li>Use 25 &mu;l of resuspended cells to inoculate 5 ml minimal media in triplicate and incubate at 37 &deg;C with shaking (300 rpm). Take OD595 readings between 15-25 h, approximately every 2 hours by transferring 200 &mu;l of each sample into a 96-well plate and reading using the plate-reader.</li>
</ol>
6. Representative Results:
An example of the use of the ToxR transcriptional reporter assay to analyze the oligomerization propensity of transmembrane domains in shown in Figure 4. Previously we have investigated the oligomerization of transmembrane domains from the multispanning membrane-integral protein latent membrane protein-1 (LMP-1) by various techniques, including ToxR.14 Transmembrane domain five (TM5) was shown to exhibit a strong propensity to oligomerize; this is demonstrated by high Miller Units, comparable to the positive control, GpA, a well-established dimerizing sequence. A deleterious mutation in TM5, D150A, reduces the ability of the sequence to oligomerize. LMP-1 TM1 does not significantly oligomerize and exhibits a very low Miller Unit signal, just above the signal for blank, non-transformed FHK12 cells.
<img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Figure 1" /> 
Figure 1. Cartoon depicting the ToxR reporter assay. Transmembrane domain (TMD) driven oligomerization results in dimerization of ToxR and activation of LacZ transcription. The gene product of LacZ, &#946;-galactosidase can be quantified as a measure of the propensity of a TMD to oligomerize.
<img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Figure 2" /> 
Figure 2. The hydrolytic cleavage of ONPG by &#946;-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP).
<img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Figure 3" /> 
Figure 3. Plasmid map of pToxR7.
<img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Figure 4" /> 
Figure 4. Representative ToxR transcriptional reporter assay analysing the oligomerization propensity of latent membrane protein-1 transmembrane domains. Transmembrane domain 5 (TM5) oligomerizes strongly, whilst transmembrane domain 1 (TM1) exhibits only a weak interaction. Mutation D150A in TM5 significantly reduces its ability to oligomerize. GpA is included as a positive control sequence for strong dimerization. Blank represents untransformed FHK12 cells.
<img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Figure 5" /> 
Figure 5. Western blot for protein expression.
<img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Figure 6" /> 
Figure 6. PD28 complementation assay to control for correct membrane insertion to the periplasm. Negative control represents a construct deficient in maltose binding protein.

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+nuclear+magnetic+resonance+structure+of+membrane-integral+diacylglycerol+kinase.">Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.</a> Science. 324, 1726-1729 (2009).</li><li>Hattori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mg(2+)-dependent+gating+of+bacterial+MgtE+channel+underlies+Mg(2+)+homeostasis.">Mg(2+)-dependent gating of bacterial MgtE channel underlies Mg(2+) homeostasis.</a> Embo J. 28, 3602-3612 (2009).</li><li>Ulmschneider, M. B., Sansom, M. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+proteins+into+membranes.">Folding proteins into membranes.</a> Nat Struct Biol. 3, 815-818 (1996).</li><li>Gerber, D., Shai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+hetero-association+of+glycophorin-A+and+its+mutants+within+the+membrane.">In vivo detection of hetero-association of glycophorin-A and its mutants within the membrane.</a> J Biol Chem. 276, 31229-31232 (2001).</li><li>Langosch, D., Brosig, B., Kolmar, H. p., Fritz, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerisation+of+the+glycophorin+A+transmembrane+segment+in+membranes+probed+with+the+ToxR+transcription+activator.">Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator.</a> J Mol Biol. 263, 525-530 (1996).</li><li>Russ, W. P., Engelman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOXCAT:+a+measure+of+transmembrane+helix+association+in+a+biological+membrane.">TOXCAT: a measure of transmembrane helix association in a biological membrane.</a> Proc Natl Acad Sci U S A. 96, 863-868 (1999).</li><li>Berger, B. 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No conflicts of interest declared.

The ToxR transcriptional reporter assay is a facile way to identify transmembrane sequences with the potential to oligomerize. Since the interactions are occurring within the bacterial inner membrane, this assay circumvents the issues associated with the validity of studying systems in membrane-mimetic environments. Given that cloning of multiple TMDs into a single plasmid can readily be done in parallel and the entire assay can be carried out in 96-well plate format, this assay can be used for high throughput analysis ...

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>BamHI restriction enzyme</td>
 <td>Invitrogen</td>
 <td>15201023</td>
 <td>Invitrogen enzymes were found to be more efficient than alternative suppliers</td>
 </tr>
 <tr>
 <td>NheI restriction enzyme</td>
 <td>Invitrogen</td>
 <td>15444011</td>
 <td>Invitrogen enzymes were found to be more efficient than alternative suppliers</td>
 </tr>
 <tr>
 <td>15 mL culture tubes</td>
 <td>Fisher Scientific</td>
 <td>14-956-1J</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SOC media</td>
 <td>Teknova</td>
 <td>S0225</td>
 <td>Made up to the appropriate volume and sterilized by autoclaving.</td>
 </tr>
 <tr>
 <td>LB media</td>
 <td>Sigma-Aldrich</td>
 <td>L7275</td>
 <td>Made up to the appropriate volume and sterilized by autoclaving.</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>CO378</td>
 <td>Stock solution of 30 mg/ mL in ethanol stored in freezer</td>
 </tr>
 <tr>
 <td>Arabinose</td>
 <td>Fluka</td>
 <td>10839</td>
 <td>Stock solution of 2.5% (w/v) in water stored in freezer</td>
 </tr>
 <tr>
 <td>Na2HPO4</td>
 <td>Sigma-Aldrich</td>
 <td>S9390</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>S9638</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Mallinckrodt Chemicals</td>
 <td>6858-06</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MgSO4.7H2O</td>
 <td>Sigma-Aldrich</td>
 <td>63138</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium dodecylsulfate (SDS)</td>
 <td>Sigma-Aldrich</td>
 <td>L6026</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-Nitrophenyl &#946;-D-galactopyranoside (ONPG)</td>
 <td>Sigma-Aldrich</td>
 <td>73660</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Z-buffer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>16.1 g Na2HPO4 
5.5g NaH2PO4 
0.75g KCl 
0.246g MgSO4 
Make up to 1 l, pH 7.0</td>
 </tr>
 <tr>
 <td>Z-buffer/chloroform</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>200 mL &#946;-mercaptoethanol, 2 mL chloroform, make up to 20 mL with Z-buffer. Vortex for 1 min, centrifuge for 1 min at 800 rpm.&nbsp; Make fresh for each plate.</td>
 </tr>
 <tr>
 <td>Z-buffer/SDS</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>160 mg SDS dissolved in 10 mL Z-buffer</td>
 </tr>
 <tr>
 <td>Z-buffer/ONPG</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>40 mg ONPG in 10 mL Z-buffer. Make fresh for each plate</td>
 </tr>
 <tr>
 <td>&#946;-mercaptoethanol</td>
 <td>Calbiochem</td>
 <td>444203</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-MBP monoclonal antibody (HRP conjugated)</td>
 <td>NEB</td>
 <td>E8038S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Minimal media with maltose</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1 x M9 salts, 0.4% maltose, 1 mg/ mL thiamin, 2 mM MgSO4</td>
 </tr>
 <tr>
 <td>96-well flat bottom plate</td>
 <td>Sarstedt</td>
 <td>83.1835.300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plate-reader</td>
 <td>Beckman Coulter</td>
 <td>DTX880 Multimode Detector</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>VWRI</td>
 <td>89032-204</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Shaking incubator</td>
 <td>FormaScientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

transmembrane domain oligomerization propensity determined by toxr assay

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.1-3 One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,8 direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.9 The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).
In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.10-13 The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where &#946;-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for &#946;-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by &#946;-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

An efficient procedure to assess the oligomerization propensity of single-pass transmembrane domains (TMDs) is described. Chimeric proteins consisting of the TMD fused to ToxR are expressed in an E. coli reporter strain. TMD-induced oligomerization causes dimerization of ToxR, activation of transcription and production of the reporter protein, -galactosidase.

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

transmembrane-domain-oligomerization-propensity-determined-toxr

Research

JoVE Journal

Biology

15.2K Aufrufe.  University of Colorado at Boulder. Ein effizientes Verfahren zur Oligomerisierung Neigung der Single-Pass-Transmembrandomänen (TMD) zu beurteilen ist beschrieben. Chimäre Proteine, bestehend aus dem TMD fusioniert ToxR sind in einer E. coli-Stamm-Reporter zum Ausdruck gebracht. TMD-induzierte Oligomerisierung Ursachen Dimerisierung von ToxR, Aktivierung der Transkription und Produktion von der Reporter-Proteins,-Galactosidase.

Serie: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...