Biz, Ulusal Sağlık Enstitüleri (1R21CA138373 teşekkür ediyor ve bu çalışmanın finansal destek Kanseri (SU2C) Stand Up. HY, Amerikan Kanser Araştırma, Sidney Kimmel Vakfı Kimmel Scholar Ödülü Derneği, 2009 Elion Ödülü için minnettar Kanser Araştırma (SKF-08-101) ve Ulusal Bilim Vakfı Fakültesi Erken Kariyer Ödülü (NSF0954819).

1. Klonlama Hususlar <ol><li> Ticari olarak hazırlanan oligonükleotidler NheI ve BamHI kısıtlama siteleri ve 5&#39;-fosforile ile çevrili ilgi TMD temsil pTox7 (BamHI kısıtlama sitesi 14 doğrudan sonra tek bir baz çifti ekleme laboratuvarımızda güncellenmiştir) (Şekil 3) sırayla sindirilir içine bağlandı . BamHI ve NheI. Bir örnek oligonükleotid, aşağıda gösterilmiştir: 5&#39;ctagcTMDSEQUENCEg3 &#39; 3 &#39;gTMDSEQUENCEcctag5&#39; </li></ol> TMD sıra 12-24 kalıntıları (kısa dizileri muhtemelen vektör kodlanmış hidrofobik artıkları uzun olacak) olmalıdır. Sıralı kalıntı eklemeler ve ToxR TMD göreli dönme birlikte kalıntı silmeler. 15,16 Nihayet, arabinoz konsantrasyonu 0.001 ve% 0.01 arasında değişmektedir olmalıdır arayüzü araştırmak için, dört değişik TMD tasarım oluşturulmalıdır (w / v) farklı TMD dizileri arasında β-galaktosidaz sinyalleri maksimum farklılıklar gözlenmektedir konsantrasyonunu belirlemek için farklı ekspresyon seviyeleri test koşulları altında farklı yakınlık en iyi ayırt edilebilir tanımlamak için tavsiye edilir. Arabinoz ve antibiyotiklere ek olarak, 0.4 mM IPTG farklı TMDS arasındaki yakınlık farkı artırmak için kullanılabilir. En az dört nüsha ToxR ölçümü yapılmalıdır. Tüm prosedürü farklı plazmid dönüşümler ile en az üç kez tekrar edilmelidir. 2. Bakteriyal Kültürler Büyüme <ol><li> Yavaşça 15 ml kültür tüp içine buz ve transfer FHK12 yetkili hücreleri (200 ul) Çözülme. Plazmid DNA (200 ng) ekleyin ve 30 dakika buz üzerinde hücreleri kuluçkaya yatmaktadır. </li><li> 42 90 s inkübasyon ısı-şok hücreleri ° C, 2 dakika süreyle buz üzerinde kuluçka takip. </li><li> SOC medya (800 ul) ekleyin ve 37 numunelerin inkübasyon ° C (300 rpm) sallayarak bir saat </li><li> Üç nüsha halinde 15 ml kültür tüpleri dönüşüm karışımı 50 ul kloramfenikol (30 mg / ml) ve arabinoz (0.0025% w / v) ile 5 ml LB medya inoküle edin. 37 numune ° C&#39;de 20 saat (300 rpm) sallayarak (5 ul kültür Alternatif 96-plaka orta 100 ul aşılamak için kullanılan olabilir, çok sayıda numune ile uğraşırken bu yöntem yararlıdır, hataları biraz daha yüksek olmasına rağmen, yol açacak buharlaşma önlemek için hata ile dış ortam kuyuları doldurup, ancak örnekler için onları kullanmayın. Sonunda), kapak ve parafilm ile plaka arasında ortak çift sarın. </li></ol> 3. Β-galaktosidaz Aktivite ölçümü <ol><li> Onceden plaka okuyucu ile 28 ° C </li><li> Üst katmanı (sulu) sadece almak için emin olun, büyük bir pipet ucu ile Z-tampon bir rezervuar içine aktarın. 96 plaka kuyulardan taze hazırlanmış Z-buffer/chloroform 100 ul aktarın. Her kültürün 5 ul dört nüsha plaka kuyuların içine aktarın. Dört kuyu boş olarak hizmet verecek kültür yok sayın. </li><li> Hücre yoğunluğu belirlemek için plaka OD 595 ölçün. </li><li> Plaka tüm kuyuları Z-buffer/SDS 50 ul ekleyin. Plaka plaka okuyucu hücreleri lyze için 10 dakika süreyle iyice çalkalayın. Hücre süspansiyonları lizis sonra açık ve gerekirse sallayarak adımı tekrarlayın emin olun. Eksik lizis Z-buffer/chloroform taze hazır değildi göstermektedir. </li><li> Tüm kuyulara Z-buffer/ONPG taze hazırlanmış 50 ul ekleyin ve 20 dakika boyunca her 30 s plaka okuyucu ve ölçü OD 405 plaka dönmek. </li><li> Aşağıdaki denklemi kullanarak (boş çıkarma hatırlamak) β-galaktosidaz aktivitesi hesaplayın. Doğrusal bir modeli uydurmak kullanarak 1,0 OD 405 aralığı 0.0 tüm veri noktalarını kullanarak, OD 405 / dk oranı hesaplanmalıdır . <img src="files/ftp_upload/2721/2721eq1.jpg" alt="Denklem 1" /> Miller birimleri, farklı günlerde bazen farklılık gösterir. Bu nedenle, genel not ortalaması gibi bir referans inşa, her bir test ölçülmelidir. Kendi değerlerini ToxR değerleri normalleşmesi için kullanılabilir. </li></ol> 4. Protein Ekspresyonu için kontrol <ol><li> Batı yapıları arasında bile protein ekspresyonu doğrulamak için blot gerçekleştirin. Bir mikrosantrifüj üç nüsha kültürler ve santrifüj 50 ul (2000 rpm, 4 dakika) birleştirin. Pipetleme süpernatantı ve 2 adet numune yükleme tamponu kalan pelet tekrar süspansiyon haline getirin. </li><li> % 8 standart bir jel üzerinde 7.5 ul Yük ve 1 saat 5 dakika boyunca 125 V electrophoreses yürütmek. Aktarımdan sonra, anti-MBP HRP-konjuge antikor ile inkübe ve görselleştirmek; kimerik protein yaklaşık 70 kDa, 48 kDa etrafında bazen görülen bazı yıkım ürünleri ile görülmektedir. Endojen MBP 45 kDa görülmektedir (bkz. Şekil 5). </li></ol>5. Uygun Membran Ekleme Kontrolü Maltoz bağlayıcı protein eksikliği hücre hattı kimerik TMD inşa uygun membran ekleme değerlendirmek için kullanılır. Tek karbon kaynağı olarak, maltoz ile minimal medya yetişen, sadece hücreleri periplasm doğru yer maltoz bağlayıcı protein ile bir zar-integral ifadesi ürün ifade büyümek mümkün. <ol><li> Transform PD28 hücreleri (FHK12 hücreleri için açıklandığı gibi) ve 2 ml LB orta aşılamak. 37 hücrelerin büyümesine ° C (300 rpm) bir gecede sallayarak. </li><li> Pelet 3500 rpm&#39;de santrifüj yoluyla hücreler, 10 dk, 4 ° C ve PBS (2 ml) tabanda büyük bir ucunda ya da yumuşak bir vorteks nazik pipetleme ile yıkayın. Pelet hücreleri (yukarıdaki gibi), ikinci kez PBS ile yıkayın pelet ve son olarak PBS (1 ml) tekrar süspansiyon haline getirin. </li><li> 37 az üç nüsha ve inkübe 5 ml minimal medya aşılamak için yeniden süspanse hücrelerinin 25 ul kullanın ° C sallayarak (300 rpm). 15-25 saat, plaka okuyucu kullanarak 96-plaka okuma ve her bir örnek 200 ul aktararak yaklaşık her 2 saat arasında OD 595 okuma alın. </li></ol> 6. Temsilcisi Sonuçlar: Şekil 4&#39;te gösterildiği transmembran etki alanı oligomerization eğilimi analiz ToxR transkripsiyonel muhabiri testinin kullanımı bir örnek. Önceleri biz oligomerization transmembran etki araştırıldı multispanning membran-integral protein latent membran protein-1 ToxR de dahil olmak üzere çeşitli teknikler (LMP-1) 14 Transmembran etki alanı beş (TM5) oligomerize güçlü bir eğilimi gösterirler gösterildi. Bu pozitif kontrol ile karşılaştırılabilecek yüksek Miller Üniteleri, GPA, köklü bir dimerizing dizisi olduğunu göstermiştir. TM5, D150A, zararlı mutasyon oligomerize dizisi yeteneğini azaltır. LMP-1 TM1 önemli ölçüde oligomerize değildir ve sadece boş olmayan dönüştürülmüş FHK12 hücreler için sinyal üzerinde çok düşük Miller Birim sinyali sergiliyor. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Şekil 1" /> Şekil 1. Karikatür ToxR muhabiri tahlil tasvir. Transmembran etki alanı (TMD) ToxR ve LacZ transkripsiyon aktivasyonu dimerization oligomerization sonuçları tahrik. LacZ gen ürünü, β-galaktosidaz oligomerize bir TMD eğilimi bir önlem olarak belirlenebilir. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Şekil 2" /> Şekil 2 ışık emici türleri o nitrophenolate (ONP) üretimi β-galaktosidaz sonuçları ONPG hidrolitik bölünme. <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Şekil 3" /> Şekil 3 pToxR7 Plazmid harita. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Şekil 4" /> Şekil 4 Temsilcisi ToxR transkripsiyonel muhabiri tahlil latent membran protein-1 transmembran etki alanı oligomerization eğilimi analiz. Transmembran etki alanı 1 (TM1) sadece zayıf bir etkileşim gösterir iken Transmembran etki alanı 5 (TM5), güçlü bir oligomerizes. Mutasyon D150A TM5 içinde oligomerize kabiliyetini önemli ölçüde azaltır. GPA, güçlü dimerization bir pozitif kontrol dizisi olarak yer almaktadır. Boş araştırıcıların bulgularıyla FHK12 hücreleri temsil eder. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Şekil 5" /> Şekil 5 protein ekspresyonu için Western Blot. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Şekil 6" /> Şekil 6 PD28 tamamlama tahlil periplasm doğru membran ekleme kontrol etmek için. Negatif kontrol, maltoz bağlayıcı protein eksikliği bir yapı gösterir.

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ToxR transkripsiyonel muhabiri tahlil oligomerize potansiyeline sahip transmembran dizileri tanımlamak için uyduruk bir yoludur. Bakteriyel iç zarı içinde meydana gelen etkileşimler olduğundan, bu testte membran-mimetik ortamlarda eğitim sistemlerinin geçerliliği ile ilgili konular circumvents. Paralel olarak tek bir plazmid birden çok TMDS klonlama kolayca yapılabilir ve tüm testi 96-plaka biçiminde yapılabilir göz önüne alındığında, bu testte yüksek verimlilik analizi için çok sayıda protein ...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> BamHI restriksiyon enzimi </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen enzimler alternatif tedarikçiler daha verimli olduğu tespit edildi </td></tr><tr><td> NheI restriksiyon enzimi </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen enzimler alternatif tedarikçiler daha verimli olduğu tespit edildi </td></tr><tr><td> 15 ml kültür tüpleri </td><td> Fisher Scientific </td><td> 14-956-1J </td><td></td></tr><tr><td> SOC medya </td><td> Teknova </td><td> S0225 </td><td> Uygun hacmi kadar yapılan ve otoklav ile sterilize. </td></tr><tr><td> LB medya </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Uygun hacmi kadar yapılan ve otoklav ile sterilize. </td></tr><tr><td> Kloramfenikol </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Dondurucuda saklanan etanol içinde 30 mg / mL stok çözelti </td></tr><tr><td> Arabinoz </td><td> Fluka </td><td> 10839 </td><td> Dondurucuda saklanan su,% 2,5 (w / v) stok solüsyonu </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> Mallinckrodt Kimyasallar </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> Sodyum dodecylsulfate (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-nitrofenil β-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 2 HPO 4 5.5g NaH 2 PO 4 0.75g KCl 0.246g MgSO 4 1 lt, pH 7.0 Makyaj </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 ml β-mercaptoethanol, 2 mL kloroform, Z-buffer ile 20 ml kadar yapmak. 800 rpm&#39;de 1 dakika için 1 dakika, santrifüj Vortex. Her plaka için taze olun. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 mg SDS 10 ml Z-tampon içinde çözünmüş </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40 mg 10 ml Z-tampon ONPG. Her plaka için taze olun </td></tr><tr><td> ß-mercaptoethanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Anti-MBP monoklonal antikor (HRP konjuge) </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td> Maltoz ile Minimal medya </td><td></td><td></td><td> 1 x M9 tuzlar,% 0.4 maltoz, 1 mg / ml tiamin, 2 mM MgSO 4 </td></tr><tr><td> 96-düz alt plakası </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Levha okuyucu </td><td> Beckman Coulter </td><td> DTX880 Multimode Dedektörü </td><td></td></tr><tr><td> Su banyosu </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Inkübatör Çalkalamalı </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

Çürütülmüştür beri protein transmembran etki basitleştirilmiş görünüm olarak sadece fosfolipid bilayers çapa uzun. Birçok durumda membran kapsayan proteinler eylem oldukça sofistike mekanizmaları gelişmiştir. Membran proteinleri, yapıları ve işlevleri modüle 1-3 hangi bir yolu yapılandırılmış transmembran oligomerler oluşturan, hidrofobik heliksler ve özel direkt temas 4,5 Kadar son tercihen sulu çözelti ve sürücü protein dernek. 6,7 Bununla birlikte, transmembran proteinlerinin etki moleküler tanıma çalışmaları hala gerisinde kalıyor bu farklı moleküller arası kuvvetler karşılaştırma membran ortamda bulunan amino asitlerin dağıtım odaklandı suda çözünen bölgeler. Büyük bir engel kalır: transmembran oligomerization elde edebilirsiniz olağanüstü spesifite ve afinite rağmen, kendi dernek 8 doğrudan ölçümü zordur. Integral membran proteini fonksiyon çalışmaya uygulanan geleneksel metodolojiler inceleme altında dizilerinin doğasında çıkılmazlık engel olabilir. Transmembran etki alanlarını temsil eden sentetik peptidlerin inceleyerek elde Biyofiziksel anlayışlar yararlı yapısal içgörü sağlayabilir. Ancak, deterjan misel veya hücre zarının taklit etmek için bu çalışmalarda kullanılan lipozom sistemleri biyolojik önemi sık sık sorgulanmaktadır; peptidler, bu koşullar altında anadili gibi bir yapı benimsiyor ve bunların fonksiyonel davranış, gerçekten yerli bir membran içinde eylem türü yansıtıyor mu ? Transmembran dizilerinin doğal fosfolipid bilayers etkileşimleri incelemek üzere, Langosch laboratuar ToxR transkripsiyonel muhabiri deneyleri geliştirdi. 9 transmembran ilgi alanı periplasm ve ToxR konumu, maltoz bağlayıcı protein ile bir kimerik protein olarak ifade edilir bir rapor sunmak oligomerization düzeyi (Şekil 1). Son on yıl içinde, diğer bazı gruplar (örneğin Engelman, DeGrado, Shai) daha da optimize ve bu ToxR muhabiri tahlil uygulanan 10-13 çeşitli ToxR deneyleri, hücre zarlarında protein-protein etkileşimleri test etmek için bir altın standart haline gelmiştir . Biz burada öncelikle Langosch tarafından geliştirilen protokoller aşağıdaki laboratuvarda yapılan tipik bir deneysel çalışma göstermektedir. Bu genellikle uygulanan yöntem E. transmembran alan öz-dernek analizi için yararlı coli, β-galaktosidaz üretim TMD oligomerization eğilimi değerlendirmek için kullanılır. TMD indüklenen dimerization üzerine, ToxR β-galaktosidaz LacZ gen up-regülasyonu neden ctx organizatörü bağlar. Kolorimetrik okuma kazandaki süreçlerin hücreleri ONPG eklenmesi ile elde edilir. Işık emici türleri o nitrophenolate (ONP) (Şekil 2) üretimini β-galaktosidaz sonuçları ONPG hidrolitik bölünme.

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

Tek-geçişli transmembran etki oligomerization eğilimi (TMDS) değerlendirmek için etkili bir prosedür açıklanmıştır. E. coli muhabiri zorlanma ToxR için erimiş TMD oluşan Kimerik proteinleri ifade edilir. TMD-kaynaklı oligomerization ToxR, transkripsiyon ve muhabir protein üretimi,-galaktosidaz aktivasyonu dimerization neden olur.

ToxR Assay tarafından belirlenen Transmembran Domain Oligomerization Eğilimi

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.2K Görüntüleme.  University of Colorado at Boulder. Tek-geçişli transmembran etki oligomerization eğilimi (TMDS) değerlendirmek için etkili bir prosedür açıklanmıştır. E. coli muhabiri zorlanma ToxR için erimiş TMD oluşan Kimerik proteinleri ifade edilir. TMD-kaynaklı oligomerization ToxR, transkripsiyon ve muhabir protein üretimi,-galaktosidaz aktivasyonu dimerization neden olur.

Video: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

Bakteriyel Konjugasyon ilgilendim Transferi Proteinlerin Dizi özgü Analiz konjugatif Çiftleşme Tahliller

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Genetik

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.

Yöntem gözünüzde canlandırın ve Transmisyon Elektron Mikroskobu Membran Etkileşen Proteinler Analiz için

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

Biyokimya

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.

Yüzey Plasmon Rezonans Host Cytosol Toksin Translokasyon Tespiti

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 ...

İmmünoloji ve Enfeksiyon

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.

Protein Korunumunun Hızlı Değerlendirilmesi için Türler Arasında Tahmin Etmek İçin Dizi Hizalamasının Gösterilmesi Duyarlılık Aracı

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

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.

X-Ray Kristalografi Thermotoga maritima M42 Aminopeptidaz TmPep1050 Oligomerik Devlet Geçiş Çalışma

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

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitokondriyal Membran Dinamiğinin Yeniden İkasini için Model Membran Platformu

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

Förster Rezonans Enerji Transfer Haritalaması: Küresel Yapısal Özellikleri Aydınlatmak İçin Yeni Bir Metodoloji

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

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.

Toplam İç Yansıma Floresan (TIRF) Mikroskobu Kullanılarak Lipid Çift Katmanlarında Yanal Mobilite ve İyon Kanalı Aktivitesinin Tek Moleküllü Görüntülenmesi

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

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Membran Proteinleri Konformasyonel Dynamics incelenmesi In situ Site Floresan Etiketleme

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Biyoloji

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Genetik ve biyokimyasal Yaklaşımlar<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Protein oligomerization Değerlendirilmesi: Riyanodin Reseptör Vaka Çalışması

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...