Jiang laboratuarında KvAP üzerinde çalışmalar Rockefeller Üniversitesi Dr Roderick MacKinnon en laboratuvarından önemli yardım almış. Özel teşekkür onların tavsiyelerini Dr Kathlynn Brown ve Michael McQuire gidip bizim faj ekran deneylere yardımcı olur. Bu çalışma NIH (Q-XJ için GM088745 ve GM093271) ve AHA (Q-XJ için 12IRG9400019) hibe tarafından desteklenmiştir.

<ol>
	<li>Alberts, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Cell. , (2007).</li><li>Lee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lipids+and+proteins+interact+in+a+membrane:+a+molecular+approach.">How lipids and proteins interact in a membrane: a molecular approach.</a> Mol. Biosyst. 1, 203 (2005).</li><li>Lee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lipids+affect+the+activities+of+integral+membrane+proteins.">How lipids affect the activities of integral membrane proteins.</a> Biochim. Biophys. Acta. 1666, 62 (2004).</li><li>Anderson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+caveolae+membrane+system.">The caveolae membrane system.</a> Annu. Rev. Biochem. 67, 199 (1998).</li><li>Simons, K., Vaz, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems,+lipid+rafts,+and+cell+membranes.">Model systems, lipid rafts, and cell membranes.</a> Annu. Rev. Biophys. Biomol. Struct. 33, 269 (2004).</li><li>Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+lipid+rafts:+from+model+membranes+to+cells.">The state of lipid rafts: from model membranes to cells.</a> Annu. Rev. Biophys. Biomol. Struct. 32, 257 (2003).</li><li>Kapoor, R., Kim, J. H., Ingolfson, H., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Artificial+Bilayers+for+Electrophysiology+Experiments.">Preparation of Artificial Bilayers for Electrophysiology Experiments.</a> J. Vis. Exp. (20), e1033 (2008).</li><li>Zheng, H., Liu, W., Anderson, L. Y., Jiang, Q. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-dependent+gating+of+a+voltage-gated+potassium+channel.">Lipid-dependent gating of a voltage-gated potassium channel.</a> Nat. Commun. 2, 250 (2011).</li><li>Schmidt, D., Jiang, Q. X., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipids+and+the+origin+of+cationic+gating+charges+in+voltage+sensors.">Phospholipids and the origin of cationic gating charges in voltage sensors.</a> Nature. 444, 775 (2006).</li><li>Ruta, V., Jiang, Y., Lee, A., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+an+archaebacterial+voltage-dependent+K++channel.">Functional analysis of an archaebacterial voltage-dependent K+ channel.</a> Nature. 422, 180 (2003).</li><li>Jiang, Q. X., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+lipids+on+voltage-gated+ion+channels.">The influence of lipids on voltage-gated ion channels.</a> Curr. Opin. Struct. Biol. , 3408884 (2012).</li><li>Artimo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ExPASy:+SIB+bioinformatics+resource+portal.">ExPASy: SIB bioinformatics resource portal.</a> Nucleic Acids Res. 40, W597 (2012).</li><li>Cladera, J., Rigaud, J. L., Villaverde, J., Dunach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+solubilization+and+membrane+protein+reconstitution+using+Chaps+and+Chapso.">Liposome solubilization and membrane protein reconstitution using Chaps and Chapso.</a> Eur. J. Biochem. 243, 798 (1997).</li><li>Levy, D., Bluzat, A., Seigneuret, M., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+study+of+liposome+and+proteoliposome+reconstitution+involving+Bio-Bead-mediated+Triton+X-100+removal.">A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal.</a> Biochim. Biophys. Acta. 1025, 179 (1990).</li><li>Young, H. S., Rigaud, J. L., Lacapere, J. J., Reddy, L. G., Stokes, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+tubular+crystals+by+reconstitution+of+detergent-solubilized+Ca2(+)-ATPase.">How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase.</a> Biophys. J. 72, 2545 (1997).</li><li>Levy, D., Gulik, A., Bluzat, A., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+sarcoplasmic+reticulum+Ca(2+)-ATPase:+mechanisms+of+membrane+protein+insertion+into+liposomes+during+reconstitution+procedures+involving+the+use+of+detergents.">Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents.</a> Biochim. Biophys. Acta. 1107, 283 (1992).</li><li>Cohen, F. S., Zimmerberg, J., Finkelstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+phospholipid+vesicles+with+planar+phospholipid+bilayer+membranes.+II.+Incorporation+of+a+vesicular+membrane+marker+into+the+planar+membrane.">Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.</a> The Journal of General Physiology. 75, 251 (1980).</li><li>Fuks, B., Homble, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+electrical+properties+of+planar+lipid+membranes+from+thylakoid+lipids.">Permeability and electrical properties of planar lipid membranes from thylakoid lipids.</a> Biophysical Journal. 66, 1404 (1994).</li><li>Hanke, W., Schlue, W. -. R., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+Lipid+Bilayers.+Methods+and+Applications.">Planar Lipid Bilayers. Methods and Applications.</a> Biological Techniques. , 133 (1993).</li><li>Tien, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bilayer lipid membranes (BLM). Theory and Practice. , 655-65 (1974).</li><li>Pagano, R. E., Ruysschaert, J. M., Miller, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+composition+of+some+lipid+bilayer+membranes+in+aqueous+solution.">The molecular composition of some lipid bilayer membranes in aqueous solution.</a> The Journal of Membrane Biology. 10, 11 (1972).</li><li>Henn, F. A., Thompson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+lipid+bilayer+membranes+separating+two+aqueous+phases:+composition+studies.">Properties of lipid bilayer membranes separating two aqueous phases: composition studies.</a> Journal of Molecular Biology. 31, 227 (1968).</li><li>Tao, X., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+Kv1.2+and+paddle+chimera+Kv+channels+in+planar+lipid+bilayers.">Functional analysis of Kv1.2 and paddle chimera Kv channels in planar lipid bilayers.</a> J. Mol. Biol. 382, 24 (2008).</li><li>Cohen, F. S., Akabas, M. H., Zimmerberg, J., Finkelstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+affecting+the+fusion+of+unilamellar+phospholipid+vesicles+with+planar+bilayer+membranes.">Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.</a> The Journal of Cell Biology. 98, 1054 (1984).</li><li>Smith, G. P., Petrenko, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Display.">Phage Display.</a> Chem. Rev. 97, 391-39 (1997).</li><li>McGuire, M. J., Li, S., Brown, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopanning+of+phage+displayed+peptide+libraries+for+the+isolation+of+cell-specific+ligands.">Biopanning of phage displayed peptide libraries for the isolation of cell-specific ligands.</a> Methods Mol. Biol. 504, 291 (2009).</li><li>Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins:+functional+and+structural+studies+using+reconstituted+proteoliposomes+and+2-D+crystals.">Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals.</a> Braz. J. Med. Biol. Res. 35, 753 (2002).</li><li>Chami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+octyl+beta-thioglucopyranoside+in+two-dimensional+crystallization+of+membrane+proteins.">Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins.</a> J. Struct. Biol. 133, 64 (2001).</li><li>Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+of+membrane+proteins.">Two-dimensional crystallization of membrane proteins.</a> Q. Rev. Biophys. 25, 1 (1992).</li><li>Rigaud, J. L., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes.">Reconstitution of membrane proteins into liposomes.</a> Methods Enzymol. 372, 65 (2003).</li><li>Walz, T., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Crystallography+of+Two-Dimensional+Crystals+of+Membrane+Proteins.">Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins.</a> J. Struct. Biol. 121 (2), 142 (1998).</li><li>Signorell, G. A., Kaufmann, T. C., Kukulski, W., Engel, A., Remigy, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+2D+crystallization+of+membrane+proteins+using+methyl-beta-cyclodextrin.">Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin.</a> J. Struct. Biol. 157, 321 (2007).</li><li>Vink, M., Derr, K., Love, J., Stokes, D. L., Ubarretxena-Belandia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+strategy+to+screen+2D+crystallization+trials+of+membrane+proteins.">A high-throughput strategy to screen 2D crystallization trials of membrane proteins.</a> J. Struct. Biol. 160, 295 (2007).</li><li>Iacovache, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2DX+robot:+a+membrane+protein+2D+crystallization+Swiss+Army+knife.">The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.</a> J. Struct. Biol. 169, 370 (2010).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Farklı membranlar içine KvAP kanalları sulandırma çeşitli çalışmalar 8-10 kullanılmıştır. Deterjan / lipid karışık miselleri ve protein / deterjan / lipid karışık misel arasında lipid dağılımı sağlanması fikri ardından, çok farklı lipid yapılmış membranlar içine KvAP yaklaşık tam sulandırma ulaşabiliyorlar. Her tetramerik KvAP kanalı tam olarak transmembran alanı kapsayacak şekilde ~ 100 lipid molekülleri ihtiyacı var. Temel gereksinim deterjan kaldırılmadan önce y...

Hücreler Döviz malzemeler ve spesifik membran proteinlerin 1 fonksiyonları yoluyla çevre ile bilgiler. Hücre zarlarında membran proteinlerinin pompalar, kanallar, reseptörler, intramembrane enzimler, bağlayıcı ve membranlar arasında yapısal destekçileri olarak işlev. Membran proteinleri etkileyen mutasyonların birçok insan hastalıkları ile ilgili olmuştur. Bu önemli ve hücre zarlarında kolayca erişilebilir olduğu için Aslında, birçok membran protein birincil ilaç hedefleri olmuştur. Bu membranlar çeşitli membran proteinlerinin yapısı ve işlevini anlamak ve mümkün insan hastalıklarda mutant proteinlerden zararlı etkileri hafifletmek için yeni yöntemler bulmak için yapmak bu nedenle çok önemlidir. Lipidler bilayers 2, 3 entegre tüm membran proteinleri çevreleyen. Ökaryotik membranlar, lipidlerin çeşitli farklı türleri microdomains 4, 5 halinde organize edilebilir olduğu bilinmektedir.Birçok zar proteinleri, bu microdomains yanı sıra membran 3, 6, hacimli sıvı faz olarak dağıtılmak üzere gösterilmiştir. Microdomains ve membran proteinlerinin teslim onlara ve bu dağılımlar fizyolojik önemi organizasyonu altında yatan mekanizma açıkça önemlidir ama tam olarak anlaşılamamıştır kalır. Membran proteinleri üzerinde lipid etkileri okuyan bir önemli teknik zorluk hemen hemen tüm yeniden proteinlerin fonksiyonel 7 böylece biyokimyasal iyi kontrollü lipid kompozisyon membranlar içine membran proteinlerinin saflaştırılmış güvenilir sulandırma olduğunu. Son birkaç yıl içinde, biz sulandırmak için A. prototip voltaj bağımlı potasyum kanal yöntemleri geliştirdi yapısal ve fonksiyonel çalışmalar için çeşitli 8-10 membran sistemleri içine pernix (KvAP). Diğerlerinden verileri ve hep birlikte lipid muhtemel voltaj algılama konformasyonel değişiklikler belirleyici olduğunu gösterdiBir voltaj kapılı bir iyon kanalı ve etki bu kanallar 11 bazı yapıları şekli olabilir. Bir sonraki, bizim yöntemleri ayrıntılı bir açıklama sağlayacak ve büyük olasılıkla bizim sonuçlarının başarılı üreme ve diğer membran proteinlerinin çalışmalara bizim yöntemlerin uzantısı sağlayacak kritik teknik ipuçları sunacak.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tryptone</td>
 <td>RPI Corp.</td>
 <td>T60060</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>RPI Corp.</td>
 <td>Y20020</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher</td>
 <td>S271-3</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>RPI Corp.</td>
 <td>T60040</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Fisher</td>
 <td>BP366-500</td>
 <td></td>
 </tr>
 <tr>
 <td>n-Dodecyl-&beta;-D-Maltoside</td>
 <td>Affymetrix</td>
 <td>D322S</td>
 <td>Sol-grade</td>
 </tr>
 <tr>
 <td>n-Octyl-&beta;-D-Glucoside </td>
 <td>Affymetrix</td>
 <td>O311</td>
 <td>Ana-grade</td>
 </tr>
 <tr>
 <td>Aprotinin</td>
 <td>RPI Corp.</td>
 <td>A20550-0.05</td>
 <td></td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>RPI Corp.</td>
 <td>L22035-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>Pepstatin A</td>
 <td>RPI Corp.</td>
 <td>P30100-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>SIGMA</td>
 <td>P7626</td>
 <td></td>
 </tr>
 <tr>
 <td>Dnase I</td>
 <td>Roche</td>
 <td>13407000</td>
 <td></td>
 </tr>
 <tr>
 <td>Bio-Bead SM-2</td>
 <td>Bio-Rad</td>
 <td>152-3920</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>RPI Corp.</td>
 <td>H75030</td>
 <td></td>
 </tr>
 <tr>
 <td>POPE</td>
 <td>Avanti Polar Lipids</td>
 <td>850757C</td>
 <td></td>
 </tr>
 <tr>
 <td>POPG</td>
 <td>Avanti Polar Lipids</td>
 <td>840457C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOGS</td>
 <td>Avanti Polar Lipids</td>
 <td>870314C</td>
 <td></td>
 </tr>
 <tr>
 <td>DMPC </td>
 <td>Avanti Polar Lipids</td>
 <td>850345C</td>
 <td></td>
 </tr>
 <tr>
 <td>Biotin-DOPE </td>
 <td>Avanti Polar Lipids</td>
 <td>870282C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOTAP</td>
 <td>Avanti Polar Lipids</td>
 <td>890890C</td>
 <td></td>
 </tr>
 <tr>
 <td>NeutrAvidin agarose beads </td>
 <td>Piercenet</td>
 <td>29200</td>
 <td></td>
 </tr>
 <tr>
 <td>Dialysis Tubing</td>
 <td>Spectrum Laboratories, Inc</td>
 <td>132-570</td>
 <td></td>
 </tr>
 <tr>
 <td>Pentane</td>
 <td>Fisher</td>
 <td>R399-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Decane</td>
 <td>TCI America</td>
 <td>D0011</td>
 <td></td>
 </tr>
 <tr>
 <td>MTS-PEG5000</td>
 <td>Toronto Research Cemicals</td>
 <td>M266501</td>
 <td></td>
 </tr>
 </tbody>
</table>

reconstitution of a kv channel into lipid membranes for structural and functional studies

Indirgeyici bir şekilde lipid protein etkileşimi araştırmak için, iyi tanımlanmış bir lipid kompozisyonu membran içine membran proteinleri de dahil için gereklidir. Biz bir prototip voltaj bağımlı potasyum (Kv) kanalında lipid bağlı yolluk etkilerini araştırıyor ve farklı membran sistemlerine sulandırmak kanallarına detaylı işlemleri çalıştım. Bizim sulandırma prosedürler deterjan indüklenen veziküllerin füzyon proteini ve / deterjan lipid / deterjan karışık miseller ile miseller gibi protein / deterjan / lipid ve deterjan arasında lipitlerin bir denge dağılımı ulaşma önemi füzyon hem de dikkate almak / lipid karışık miseller. Bizim veri lipid veziküller içinde kanallarının ekleme yönelimleri nispeten rastgele önerdi ve sulandırma verimliliği saptanabilir protein agrega fraksiyon deneylerde görüldü o kadar yüksektir. Biz sulandırmak kullanmış olmasıFarklı lipid kanalların yapısal durumları belirlemek için d kanalları, düzlemsel çift katlı lipit katmanını dahil kanalları az sayıda, bir faj görüntülenir peptid kütüphane uyum özgü ligandlar için ekranın elektrik faaliyetleri kayıt ve 2D kristallerin büyümesini desteklemek zarlarında kanalları. Burada anlatılan sulandırılması prosedürleri, özellikle ökaryotik voltaj kapılı iyon kanalları lipid etkilerinin araştırılması için, lipid tabakalarıdır Diğer zar proteinleri çalışmak için adapte edilebilir.

Biyokimyasal homojenlik içine KvAP kanalın üretilmesi için deneyler genel akışı Şekil 1A tarif edilmektedir. Proteinin ekspresyonu ve saflaştırılması sırasında tipik örnekleri, Şekil 1B &#39;de SDS-PAGE jel gösterdi. IMAC saflaştırmadan sonra proteinin nispeten saf. KvAP kanalın verimi yaklaşık 1.0 mg / litre cinsinden bir kültürdür. Deterjanlarla lipid veziküllerin çözünmüş lipid vs deterjan her çift için çok çaba harcanma...

Watch this Scientific Journal Video about Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies at JoVE.com

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Lipid membranlar bir prototip voltaj kapılı potasyum kanalı tam sulandırılması için prosedürler açıklanmıştır. Sulandırılmış kanallar biyokimyasal testler, elektrik kayıtları, ligand ve tarama elektron kristalografik çalışmalar için uygundur. Bu yöntemler, başka zar proteinlerinin yapısal ve fonksiyonel çalışmalar için genel uygulamalar olabilir.

Yapısal ve İşlevsel Araştırmalar için Lipid Membranlar bir Kv Kanal Sulandırma

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

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Biology

19.3K Görüntüleme.  University of Texas Southwestern Medical Center at Dallas. Lipid membranlar bir prototip voltaj kapılı potasyum kanalı tam sulandırılması için prosedürler açıklanmıştır. Sulandırılmış kanallar biyokimyasal testler, elektrik kayıtları, ligand ve tarama elektron kristalografik çalışmalar için uygundur. Bu yöntemler, başka zar proteinlerinin yapısal ve fonksiyonel çalışmalar için genel uygulamalar olabilir.

Video: Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidik Mesophases kullanarak Yapı Tayini için Membran Proteinleri Kristalize

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Biyoloji

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Elektron Kristalografi Yapısal Biyoloji Araştırmaları Küçük Membran Proteinleri İki boyutlu Kristalizasyon Denemeler değerlendirilmesi

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Fonksiyonel ve Yapısal Analizler için Peptidil tRNAs İçeren Çeviri Ribozomlar izolasyonu

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

Nanodisc Lipid Parçacıklar içine bir ABC Transporter Sulandırma için bir adım-adım yöntemi

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidik Mesophases Membran Proteinleri Yüksek throughput Kristalizasyon için bir robot kullanımı

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Makromoleküler Kristalografi tarafından Yapı Tayini için lipidik Mesophases Yetiştirilen Membran Proteinlerinin Hasat ve Cryo-soğutma Kristaller

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Mürin Atriyal ve Ventriküler kardiyomiyositlerde izolasyonu ve Kv Kanal Kayıtları

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Verimli Üretimi ve Saflaştırılması Rekombinant murin kindlin-3 Biophysical Çalışmaları Böcek hücrelerinden

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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.

Bir transmembran proteinin yeniden oluşturulması, Mikroskop ve yama kelepçe Çalışmaları Dev tek-katmanlı keseler içine Voltaj kapılı iyon kanalı, KvAP,

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