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

Мы благодарим Национальных Институтов Здоровья (1R21CA138373 и резервных до рака (SU2C) за финансовую поддержку этой работы. HY благодарен за 2009 Elion награду от Американской ассоциации исследований рака, премии Kimmel ученый из Сидни Киммель фонда Исследования Рака (SKF-08-101) и Национального научного фонда факультета Рано премии Карьера (NSF0954819).

1. Клонирование Соображения <ol><li> Коммерчески подготовленные олигонуклеотиды представляющих интерес TMD окружении NheI и сайтах BamHI ограничения и 5&#39;-фосфорилированных можно лигируют pTox7 (изменение в нашей лаборатории вставки одной пары оснований непосредственно после сайта BamHI огранич...

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	<li>Parker, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+heptahelical+G+protein+coupling+receptors:+a+case+for+association+using+transmembrane+helices.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+human+ATP-binding+cassette+transporters+and+its+potential+significance+in+human+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-protein+topology.">Membrane-protein topology.</a> Nat Rev Mol Cell Biol. 7, 909-918 (2006).</li><li>Van Horn, W. 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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>

ToxR транскрипционного анализа репортер легкий способ идентифицировать трансмембранного последовательностей с потенциалом oligomerize. Поскольку взаимодействия, происходящие в бактериальной внутренней мембраной, этот анализ позволяет обойти проблемы, связанные с изучением действия сист...

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии (необязательно) </th></tr><tr><td> BamHI рестрикции </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen ферменты оказались более эффективными, чем альтернативных поставщиков </td></tr><tr><td> NheI рестрикции </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen ферменты оказались более эффективными, чем альтернативных поставщиков </td></tr><tr><td> 15 мл труб культуры </td><td> Fisher Scientific </td><td> 14-956-1J </td><td></td></tr><tr><td> SOC СМИ </td><td> Teknova </td><td> S0225 </td><td> Сделано до соответствующего объема и стерилизовать в автоклаве. </td></tr><tr><td> LB СМИ </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Сделано до соответствующего объема и стерилизовать в автоклаве. </td></tr><tr><td> Хлорамфеникол </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Маточного раствора 30 мг / мл в этаноле, хранящиеся в морозильнике </td></tr><tr><td> Арабинозы </td><td> Fluka </td><td> 10839 </td><td> Маточный раствор 2,5% (м / о) в воде хранится в морозильной камере </td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9390 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma-Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Химические вещества Mallinckrodt </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> Sigma-Aldrich </td><td> 63138 </td><td></td></tr><tr><td> Додецилсульфата натрия (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-нитрофенил β-D-галактопиранозид (ONPG) </td><td> Sigma-Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-буфера </td><td></td><td></td><td> 16,1 г Na 2 HPO 4 5,5 г NaH 2 PO 4 0.75g KCl 0.246g MgSO 4 Сделать до 1 л, рН 7,0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 мл β-меркаптоэтанол, 2 мл хлороформа, составляют до 20 мл с Z-буфера. Vortex в течение 1 мин, центрифуги в течение 1 мин при 800 оборотах в минуту. Сделать свежее для каждой пластины. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 мг SDS растворяют в 10 мл Z-буфера </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40 мг ONPG в 10 мл Z-буфера. Сделать свежее для каждой пластины </td></tr><tr><td> β-меркаптоэтанол </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Анти-MBP моноклональное антитело (HRP сопряженных) </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td> Минимальная сред с мальтозы </td><td></td><td></td><td> 1 х M9 соли, 0,4% мальтозы, 1 мг / мл, тиамин, 2 мМ MgSO 4 </td></tr><tr><td> 96-луночного плоской пластине нижней </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Пластина-ридер </td><td> Beckman Coulter </td><td> DTX880 Многомодовые детектор </td><td></td></tr><tr><td> Водяная баня </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Shaking инкубатор </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

Эффективная процедура для оценки олигомеризации склонность однопроходный трансмембранных доменов (TMDS) описывается. Химерные белки, состоящие из TMD слит с ToxR выражаются в репортеру штамма E.coli. TMD-индуцированной олигомеризации причины димеризации ToxR, активации транскрипции и производства репортер белка,-галактозидазы.

Трансмембранного домена олигомеризации склонность определяется Пробирной ToxR

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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