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 β-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 μl) 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 °C, followed by incubation on ice for 2 min.</li>
 <li>Add SOC media (800 μl) and incubate the samples at 37 °C with shaking (300 rpm) for one h.</li>
 <li>Inoculate 5 ml LB media with chloramphenicol (30 μg/ml) and arabinose (0.0025% w/v) with 50 μl of the transformation mixture in 15 ml culture tubes in triplicate. Incubate samples at 37 °C with shaking (300 rpm) for 20 h. (Alternatively 5 μl of culture can be used to inoculate 100 μl 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 β-galactosidase Activity
<ol>
 <li>Preheat the plate-reader to 28 °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 μl of freshly prepared Z-buffer/chloroform to the wells of a 96-well plate. Transfer 5 μl 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 μl 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 μl 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 β-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="Miller Units formula; equation for enzymatic activity; OD405/OD595; scientific analysis." data-altupdatedat="1747911167000" data-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 μl 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 μl 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 °C with shaking (300 rpm) overnight.</li>
 <li>Pellet the cells by centrifugation at 3500 rpm, 10 min, 4 °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 μl of resuspended cells to inoculate 5 ml minimal media in triplicate and incubate at 37 °C with shaking (300 rpm). Take OD595 readings between 15-25 h, approximately every 2 hours by transferring 200 μl 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="Bacterial protein transport diagram; ToxR signaling, LacZ transcription regulation mechanism." data-altupdatedat="1747911167000" data-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, β-galactosidase can be quantified as a measure of the propensity of a TMD to oligomerize.
<img src="/files/ftp_upload/2721/2721fig2.jpg" alt="ONPG hydrolysis reaction diagram, β-galactosidase, molecular structure, lactose analog, color change." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. The hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP).
<img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Plasmid map diagram for pToxR7; features origins, promoters, markers, restriction sites." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Plasmid map of pToxR7.
<img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Bar chart showing Miller units for TM5, TM5 D150A, TM1, GpA, Blank; enzyme activity analysis." data-altupdatedat="1747911167000" data-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="Protein electrophoresis gel displaying chimeric protein and natural MBP bands for comparison." data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Western blot for protein expression.
<img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Microbial growth curve, OD595 vs. Time; line graph showing pTox7 variants and negative control." data-altupdatedat="1747911167000" data-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.

<ol><li>Parker, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligomerization+of+the+heptahelical+G+protein+coupling+receptors%3A+a+case+for+association+using+transmembrane+helices%5BTitle%5D)+AND+%22Mini+Rev+Med+Chem%22%5BJournal%5D)">Oligomerization of the heptahelical G protein coupling receptors: a case for association using transmembrane helices.</a> Mini Rev Med Chem. 9, 329-339 (2009).</li>
<li>Mo, W., Zhang, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligomerization+of+human+ATP-binding+cassette+transporters+and+its+potential+significance+in+human+disease%5BTitle%5D)+AND+%22Expert+Opin+Drug+Metab+Toxicol%22%5BJournal%5D)">Oligomerization of human ATP-binding cassette transporters and its potential significance in human disease.</a> Expert Opin Drug Metab Toxicol. 5, 1049-1063 (2009).</li>
<li>von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane-protein+topology%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Membrane-protein topology.</a> Nat Rev Mol Cell Biol. 7, 909-918 (2006).</li>
<li>Van Horn, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solution+nuclear+magnetic+resonance+structure+of+membrane-integral+diacylglycerol+kinase%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Mg%282%2B%29-dependent+gating+of+bacterial+MgtE+channel+underlies+Mg%282%2B%29+homeostasis%5BTitle%5D)+AND+%22Embo+J%22%5BJournal%5D)">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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amino+acid+distributions+in+integral+membrane+protein+structures%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Amino acid distributions in integral membrane protein structures.</a> Biochim Biophys Acta. 1512, 1-14 (2001).</li>
<li>Deber, C. M., Goto, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Folding+proteins+into+membranes%5BTitle%5D)+AND+%22Nat+Struct+Biol%22%5BJournal%5D)">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/pubmed?term=((In+vivo+detection+of+hetero-association+of+glycophorin-A+and+its+mutants+within+the+membrane%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Dimerisation+of+the+glycophorin+A+transmembrane+segment+in+membranes+probed+with+the+ToxR+transcription+activator%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((TOXCAT%3A+a+measure+of+transmembrane+helix+association+in+a+biological+membrane%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Consensus+motif+for+integrin+transmembrane+helix+association%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Strong+oligomerization+behavior+of+PDGFbeta+receptor+transmembrane+domain+and+its+regulation+by+the+juxtamembrane+regions%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">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/pubmed?term=((Hetero-assembly+between+all-L-+and+all-D-amino+acid+transmembrane+domains%3A+forces+involved+and+implication+for+inactivation+of+membrane+proteins%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Transmembrane+peptides+used+to+investigate+the+homo-oligomeric+interface+and+binding+hot-spot+of+the+oncogene%2C+latent+membrane+protein+1%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Transmembrane peptides used to investigate the homo-oligomeric interface and binding hot-spot of the oncogene, latent membrane protein 1.</a> , Forthcoming (2010).</li>
<li>Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dimerization+of+the+transmembrane+domain+of+Integrin+alphaIIb+subunit+in+cell+membranes%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((The+interface+between+self-assembling+erythropoietin+receptor+transmembrane+segments+corresponds+to+a+membrane-spanning+leucine+zipper%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((The+single+transmembrane+domains+of+human+receptor+tyrosine+kinases+encode+self-interactions%5BTitle%5D)+AND+%22Sci+Signal%22%5BJournal%5D)">The single transmembrane domains of human receptor tyrosine kinases encode self-interactions.</a> Sci Signal. 2, ra56-ra56 (2009).</li>
</ol>

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><tbody><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></td></tr><tr><td>SOC media</td><td>TEKnova, Inc.</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>CO&lt;sub&gt;3&lt;/sub&gt;78</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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9390</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>KCl</td><td>Mallinckrodt Baker Inc.</td><td>6858-06</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>63138</td><td></td></tr><tr><td>Sodium dodecylsulfate (SDS)</td><td>Sigma-Aldrich</td><td>L6026</td><td></td></tr><tr><td>2-Nitrophenyl &amp;#946;-D-galactopyranoside (ONPG)</td><td>Sigma-Aldrich</td><td>73660</td><td></td></tr><tr><td>Z-buffer</td><td></td><td></td><td>16.1 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt;5.5g NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt;0.75g KCl&lt;br/&gt;0.246g MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt;Make up to 1 l, pH 7.0</td></tr><tr><td>Z-buffer/chloroform</td><td></td><td></td><td>200 mL &amp;#946;-mercapt&amp;#8211;thanol, 2 mL chloroform, make up to 20 mL with Z-buffer. Vortex for 1 min, centrifuge for 1 min at 800 rpm. Make fresh for each plate.</td></tr><tr><td>Z-buffer/SDS</td><td></td><td></td><td>160 mg SDS dissolved in 10 mL Z-buffer</td></tr><tr><td>Z-buffer/ONPG</td><td></td><td></td><td>40 mg ONPG in 10 mL Z-buffer. Make fresh for each plate</td></tr><tr><td>&amp;#946;-mercapt&amp;#8211;thanol</td><td>Calbiochem</td><td>444203</td><td></td></tr><tr><td>Anti-MBP monoclonal antibody (HRP conjugated)</td><td>New England Biolabs</td><td>E8038S</td><td></td></tr><tr><td>Minimal media with maltose</td><td></td><td></td><td>1 x M9 salts, 0.4% maltose, 1 mg/ mL thiamin, 2 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>96-well flat bottom plate</td><td>Sarstedt Ltd</td><td>83.1835.300</td><td></td></tr><tr><td>Plate-reader</td><td>Beckman Coulter Inc.</td><td>DTX880 Multimode Detector</td><td></td></tr><tr><td>Water bath</td><td>VWR international</td><td>89032-204</td><td></td></tr><tr><td>Shaking incubator</td><td>Forma Scientific</td><td></td><td></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 Views.  University of Colorado at Boulder. Een efficiënte procedure om de oligomerisatie neiging van single-pass transmembraan-domeinen (TMDs) te beoordelen wordt beschreven. Chimère eiwitten bestaande uit de TMD gefuseerd aan ToxR worden uitgedrukt in een E. coli stam verslaggever. TMD-geïnduceerde oligomerisatie oorzaken dimerisatie van ToxR, activatie van transcriptie en de productie van de reporter eiwit-galactosidase.

Video: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

Chemistry

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biochemistry

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

Immunology and Infection

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online screening application that allows researchers and regulators to extrapolate toxicity information across species. For biological targets in model systems such as human cells, mice, rats, and zebrafish, toxicity data are available for a variety of chemicals. Through the evaluation of protein target conservation, this tool can be used to extrapolate data generated from such model systems to thousands of other species lacking toxicity data, yielding predictions of relative intrinsic chemical susceptibility. The latest releases of the tool (versions 2.0-6.1) have incorporated new features that allow for the rapid synthesis, interpretation, and use of the data for publication plus presentation-quality graphics.
Among these features are customizable data visualizations and a comprehensive summary report designed to summarize SeqAPASS data for ease of interpretation. This paper describes the protocol to guide users through submitting jobs, navigating the various levels of protein sequence comparisons, and interpreting and displaying the resulting data. New features of SeqAPASS v2.0-6.0 are highlighted. Furthermore, two use-cases focused on transthyretin and opioid receptor protein conservation using this tool are described. Finally, SeqAPASS&#39; strengths and limitations are discussed to define the domain of applicability for the tool and highlight different applications for cross-species extrapolation.

Here, we present a protocol to utilize the latest version of the US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. This protocol demonstrates the application of the online tool to rapidly analyze protein conservation and provide customizable and easily interpretable predictions of chemical susceptibility across species.

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online ...

Genetics

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

Monitoring GTPase Atlastin in Different Nucleotide Loading States

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver of homotypic membrane fusion within the endoplasmic reticulum (ER). Disruptions in this process have been associated with hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Structural and biochemical studies suggest that the conformational changes in ATL are linked to GTP hydrolysis, but real-time visualization of these conformational dynamics during the GTP hydrolysis cycle remains challenging. To better understand the mechanical mechanisms behind ATL function, single-molecule F&#246;rster resonance energy transfer (smFRET) was utilized. Three specific strategies were employed to immobilize the N-terminal cytosolic region of human ATL1 (ATL1cyto) in a streptavidin-coated microfluidic chamber, facilitating the application of intramolecular and intermolecular smFRET imaging. This allowed precise monitoring of protein conformations in various nucleotide-loading states, providing direct insights into individual molecular behaviors. This method can be applied to study other mechanochemical proteins as well.

The functions of dynamin superfamily proteins depend on conformational changes coupled with GTP hydrolysis. A system is described using the single-molecule FRET (smFRET) technique to monitor the conformational dynamics of dynamin-like GTPase atlastin in different nucleotide-loading states.

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major,&#160;offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While ...

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Samenvatting

Meer video's verkennen