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

हम स्वास्थ्य के राष्ट्रीय संस्थान (1R21CA138373 धन्यवाद और इस काम के वित्तीय समर्थन के लिए कैंसर (SU2C) करने के लिए स्टैंड अप हरियाणा के लिए कैंसर, Sidney Kimmel फाउंडेशन से एक Kimmel विद्वान पुरस्कार अनुसंधान के अमेरिकन एसोसिएशन से 2009 Elion पुरस्कार के लिए आभारी है. कैंसर रिसर्च (SKF-08-101), और राष्ट्रीय विज्ञान फाउंडेशन संकाय जल्दी कैरियर पुरस्कार (NSF0954819).

1. क्लोनिंग संबंधी बातें <ol><li> व्यावसायिक तौर पर तैयार NheI और BamHI प्रतिबंध साइटों और 5&#39;-phosphorylated द्वारा flanked ब्याज की TMD प्रतिनिधित्व oligonucleotides pTox7 (एक आधार जोड़ी के सम्मिलन से हमारी प्रयोगशाला में BamHI प्रतिबंध 14 सा?...

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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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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>

ToxR transcriptional संवाददाता परख सतही oligomerize क्षमता के साथ transmembrane दृश्यों की पहचान का तरीका है. चूंकि बातचीत बैक्टीरियल भीतरी झिल्ली के भीतर उत्पन्न कर रहे हैं, इस परख झिल्ली अनुकरण करनेवाला वातावरण में अध्ययन प्रण?...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियां (वैकल्पिक) </th></tr><tr><td> BamHI प्रतिबंध एंजाइम </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen एंजाइमों के लिए वैकल्पिक आपूर्तिकर्ताओं से अधिक कुशल होना पाया गया </td></tr><tr><td> NheI प्रतिबंध एंजाइम </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen एंजाइमों के लिए वैकल्पिक आपूर्तिकर्ताओं से अधिक कुशल होना पाया गया </td></tr><tr><td> 15 एमएल संस्कृति ट्यूबों </td><td> फिशर साइंटिफिक </td><td> 14-956-1J </td><td></td></tr><tr><td> समाज मीडिया </td><td> Teknova </td><td> S0225 </td><td> अप करने के लिए उचित मात्रा मेड और autoclaving द्वारा निष्फल. </td></tr><tr><td> लेग मीडिया </td><td> सिग्मा Aldrich </td><td> L7275 </td><td> अप करने के लिए उचित मात्रा मेड और autoclaving द्वारा निष्फल. </td></tr><tr><td> Chloramphenicol </td><td> सिग्मा Aldrich </td><td> 3 78 सीओ </td><td> इथेनॉल में 30 मिलीग्राम / एमएल के स्टॉक समाधान फ्रीजर में संग्रहीत </td></tr><tr><td> Arabinose </td><td> Fluka </td><td> 10839 </td><td> 2.5% के शेयर पानी में (w / v) समाधान फ्रीजर में संग्रहीत </td></tr><tr><td> ना दो चार HPO </td><td> सिग्मा Aldrich </td><td> S9390 </td><td></td></tr><tr><td> नाह 4 पीओ 2 </td><td> सिग्मा Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Mallinckrodt रसायन </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 0.7 एच 2 हे </td><td> सिग्मा Aldrich </td><td> 63138 </td><td></td></tr><tr><td> सोडियम dodecylsulfate (एसडीएस) </td><td> सिग्मा Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2 Nitrophenyl β डी galactopyranoside (ONPG) </td><td> सिग्मा Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-बफर </td><td></td><td></td><td> 16.1 छ ना 2 4 HPO 5.5g नाह 4 पीओ 2 0.75g KCl 0.246g 4 MgSO 1 एल, पीएच 7.0 अप करें </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 एमएल β-mercaptoethanol, 2 एमएल क्लोरोफॉर्म, 20 एमएल के लिए z-बफर के साथ. 800 rpm पर 1 मिनट के लिए 1 मिनट अपकेंद्रित्र, भंवर. एक थाली के लिए ताजा करें. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 मिग्रा एसडीएस 10 एमएल Z-बफर में भंग </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 10 एमएल Z-बफर में 40 मिलीग्राम ONPG. प्रत्येक प्लेट के लिए ताजा बनाओ </td></tr><tr><td> β-mercaptoethanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> विरोधी MBP मोनोक्लोनल एंटीबॉडी (संयुग्मित एचआरपी) </td><td> ठोंग </td><td> E8038S </td><td></td></tr><tr><td> Maltose साथ मिनिमल मीडिया </td><td></td><td></td><td> 1 x M9 लवण, 0.4% maltose, 1 मिलीग्राम / एमएल thiamin, 2 मिमी MgSO 4 </td></tr><tr><td> 96 अच्छी तरह से फ्लैट नीचे की थाली </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> थाली पाठक </td><td> Beckman कल्टर </td><td> DTX880 बहुपद्वति वेक्षक </td><td></td></tr><tr><td> जल स्नान </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> इनक्यूबेटर हिलती </td><td> FormaScientific </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

एकल पास transmembrane डोमेन की oligomerization प्रवृत्ति (TMDs) का आकलन करने के लिए एक कुशल प्रक्रिया में वर्णित है. Chimeric ToxR के लिए इनकार TMD से मिलकर प्रोटीन ई. कोलाई संवाददाता तनाव में व्यक्त कर रहे हैं. TMD प्रेरित oligomerization ToxR, प्रतिलेखन और रिपोर्टर प्रोटीन के उत्पादन, galactosidase के सक्रियण के dimerization का कारण बनता है.

Transmembrane डोमेन Oligomerization प्रवृत्ति ToxR परख द्वारा निर्धारित

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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