Name: transmembrane domain oligomerization propensity determined by toxr assay
Uploaded: 2011-05-26T14:00:00
Description: Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

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 s...

<ol>
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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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+motif+for+integrin+transmembrane+helix+association.">Consensus motif for integrin transmembrane helix association.</a> Proc Natl Acad Sci U S A. 107, 703-708 (2010).</li><li>Oates, J., King, G., Dixon, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+oligomerization+behavior+of+PDGFbeta+receptor+transmembrane+domain+and+its+regulation+by+the+juxtamembrane+regions.">Strong oligomerization behavior of PDGFbeta receptor transmembrane domain and its regulation by the juxtamembrane regions.</a> Biochim Biophys Acta. 1798, 605-615 (2010).</li><li>Sal-Man, N., 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=Hetero-assembly+between+all-L-+and+all-D-amino+acid+transmembrane+domains:+forces+involved+and+implication+for+inactivation+of+membrane+proteins.">Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins.</a> J Mol Biol. 344, 855-864 (2004).</li><li>Sammond, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+peptides+used+to+investigate+the+homo-oligomeric+interface+and+binding+hot-spot+of+the+oncogene,+latent+membrane+protein+1.">Transmembrane peptides used to investigate the homo-oligomeric interface and binding hot-spot of the oncogene, latent membrane protein 1.</a> , (2010).</li><li>Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization+of+the+transmembrane+domain+of+Integrin+alphaIIb+subunit+in+cell+membranes.">Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes.</a> J Biol Chem. 279, 26666-26673 (2004).</li><li>Ruan, W., Becker, V., Klingmuller, U., Langosch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interface+between+self-assembling+erythropoietin+receptor+transmembrane+segments+corresponds+to+a+membrane-spanning+leucine+zipper.">The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper.</a> J Biol Chem. 279, 3273-3279 (2004).</li><li>Finger, C., Escher, C., Schneider, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single+transmembrane+domains+of+human+receptor+tyrosine+kinases+encode+self-interactions.">The single transmembrane domains of human receptor tyrosine kinases encode self-interactions.</a> Sci Signal. 2, ra56-ra56 (2009).</li></ol>

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 ...

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