Name: membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution
Uploaded: 2016-08-16T13:00:00.000+00:00
Duration: 11 min 55 s
Description: Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

We thank Barbara Windschiegl for her help in establishing SOPs; Dennis Remme for his work on the NanoCalcFX software and Alina Kollmannsperger, Markus Braner and Milan Gerovac for helpful suggestions on the manuscript. The German-Israeli Project Cooperation (DIP) provided by the DFG and the Federal Ministry of Education and Research to R.T., as well as the Federal Ministry of Economics and Technology (ZIM R&amp;D Project) to R.T. and Nanospot GmbH supported this work.

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The authors declare no competing financial interests.

Burada sunulan teknik membran proteini ulaşım son derece paralel analiz sağlar. Sulandırılmış membran proteini sistemleri doğrudan teorik her zar taşıyıcı uyarlanmasını yapmadan veya olası kanala, Biochip uygulanabilir. Taşıma analizi sadece doğrudan floresans değişimi (fluorophores translokasyon veya floresan etiketli substratlar) ya da dolaylı olarak floresans değişimi (pH&#39;a duyarlı boyalar, sekonder enzimatik reaksiyonlar) yoluyla, bir flüoresan okuması sisteminin kurulması ile sınırl...

membran proteinlerinin analizi son 20 yılda temel ve ilaç araştırmaları için artan ilgi haline gelmiştir. yeni ilaçların geliştirilmesi halen kısıtlayan faktörlerden biri olan tanımlanması ve yeni hedefler detaylı karakterizasyonu bağlıdır. Tüm ilaç hedefleri yaklaşık% 60 zar proteinleri 1 olması, tekniklerin geliştirilmesi fonksiyonları önemli aydınlatmak için yapar. 4 - Geçmişte, electrogenic kanalları ve taşıyıcıları çalışma teknikleri 2 sayıda geliştirilmiştir. Aksine olmayan electrogenic yüzeyler daha zorlu bir görev sunuyoruz. Onlar kaskadlar 5 sinyal kilit reseptörler olarak hücre zarı ve fonksiyonu üzerinde çözünen ve besin akışını kontrol gibi onlar, ancak ana ilaç hedefleri olarak özel bir öneme sahiptir. Önemli çaba t gelişimi girmiştirtekniki membran transport proteinleri 6, 7 işlevini incelemek için. Birkaç isim katı desteklenen lipid bilayers, gergin bilayers 11, 12, microblack lipit membranlar 13, 14 ve yerli vezikül dizileri 15, 16 olmak üzere 10, - Katı destekli membranlar kullanılarak Sistemleri bu alanda 8 olarak en umut verici araçlar ortaya çıkmıştır. Bazıları ticari kurulumları 17, 18 hatta mevcuttur. Bazı örnekler son derece paralel bir şekilde 14, 19, tarama uygulamaları için bir ön koşul olarak, tek zar proteinleri çalışma yeteneği birleştiren yayınlanmıştır. Ancak, bu yöntemler nadiren endüstriyel ortamlarda temel araştırma köprü. güçlükler çoğu otomatikleştirilebilir için sistemin özelliği, yüksek maliyetli üretim ve / veya zahmetli hazırlanmasında uzanmaktadır. Bir yaklaşım oYukarıda açıklanan tüm engelleri aşılabilir nihai amaçtır. 22 - Burada sunulan teknik tek bir protein düzeyinde 20 kontrollü bir ortamda in vitro membran kanalları ve taşıyıcıları incelemek için geliştirilmiştir. 26 ya da siyah lipit membranlar 27 - luvs içine saflaştırılmış membran proteinlerinin Sulandırma daha GUVs 23 karşılaştırılabilir yaklaşımlara göre kurulmuştur. Direkt olarak iki katmanlı oluşum şeklinde kendini montaj işlemi ile gerçekleşiyor yonga yüzeyi, uygulanabilir. Nano-gözenekli çip (Şek. 1) ve cam alt tasarım sistemi basit otomasyon izin hava mikroskopi için izin verir. motorlu bir sahne ile birlikte çoklu cips analiz için mühürlü boşlukların binlerce içeren görüş her alanı ile aynı anda ölçülebilir. Şekil 1. Çoklanmış nanopore bioçipler tasarımı. A) silikon üzerinde yalıtkan (SOI) gofret reaktif iyon dağlama ile yapılandırılmıştır. Yaklaşık 1,150 ayrı cips aynı özellikleri ve kalite. B) Her çip nano delikler ile 250.000 bireysel, mikro içermektedir her gofret imal edilmektedir. Ölçek çubuğu:. 200 mikron C) Her oyuk multi-spektral floresan okuma-out üzerinden adreslenebilir. Ters floresan mikroskobu. D) Atomik kuvvet mikroskobu ile biyoçip uyumlu hale bir şeffaf üst tabaka blokları tampon hazneden floresan sinyalleri (AFM) görüntüleme eşit (gözenek açıklıkları ve 3.6 nm silikon dioksit tabakasının yüzey pürüzlülüğü düzenlenmiş ortaya koymaktadır n = vezikül füzyon 40) optimum. Ölçek çubuğu:. 5 mikron E) Taramalı elektron mikrofonfloroskop (SEM) görüntüsü silikon çip içinde femtoliter boşluklara erişim sağlayan nano-gözeneklere enine kesitini göstermektedir. Bu rakam 21 izni ile yeniden yapıldı. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Tüm veri analizi, son kullanıcılar için sınırsız erişim sağlamak için ücretsiz kullanarak gerçekleştirilir. Zaman serisi ücretsiz görüntü işleme yazılımları ve özel bir yapı eğrisi analiz yazılımı sağlayan toplu işleme ve eğrilerin çoklu floresan kanalları ve binlerce büyük veri kümelerinin basit korelasyon kullanılarak analiz edilmektedir. Bu protokolde kullanılan model proteini E. türetilen büyük iletkenlik (MSCL) kanal proteinin mechanosensitive kanal E. coli. Doğada ozmotik şok serbest bırakmak için bir valf olarak davranır, fakat rasyonel SYNT tasarlanmış bir şekilde değiştirildiinfographie işlevleri kovalent kanal daralma tarafına monte edilebilir. Nano-valf oluşturarak kanal açmak için tetiklenir kovalent bağlı aktivatör (MTSET) şarj-itme Via. iyonları, su, küçük proteinler, aynı zamanda küçük fluorophores gibi küçük moleküller kanalından nüfuz edebilir. Burada, bir protein, protein aracılı translokasyon algılamak üzere yeteneğini göstermek için bir model olarak kullanılır.

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>[2-(Trimethylammonium)ethyl]&lt;br/&gt; methanethiosulfonate</td><td>Toronto Research Chemicals Inc.</td><td>T792900</td><td>MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 &amp;deg;C. Use immediately (30 minutes) after solubilization in buffer.</td></tr><tr><td>1 ml gas-tight syringe</td><td>Hamilton</td><td>#1001</td><td></td></tr><tr><td>10 ml round flask</td><td>Schott Duran</td><td></td><td></td></tr><tr><td>2.7 mm glas beads</td><td>Roth</td><td>N032.1</td><td></td></tr><tr><td>2-Propanole</td><td>Roth</td><td>9866.5</td><td></td></tr><tr><td>30 cm Luer-Lock Extension Tube</td><td>Sarstedt</td><td>744304</td><td></td></tr><tr><td>Acetone</td><td>Roth</td><td>5025.5</td><td></td></tr><tr><td>Bio-Beads SM-2 Adsorbent</td><td>Bio-Rad</td><td>152-3920</td><td>need to be activated before first use</td></tr><tr><td>CaCl&lt;sub&gt;2 &lt;/sub&gt;Dihydrat</td><td>Roth</td><td>HN04.3</td><td></td></tr><tr><td>Calcein</td><td>Sigma</td><td>C0875</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Chloroform reagent grade</td><td>VWR Chemicals</td><td>22711324</td><td></td></tr><tr><td>DOPE&lt;sup&gt;ATTO390&lt;/sup&gt;</td><td>ATTO-TEC</td><td>AD 390-165</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Ethanol absolute</td><td>Sigma-Aldrich</td><td>32205</td><td></td></tr><tr><td>Injekt Single-use syringe</td><td>Braun</td><td>460 60 51V</td><td></td></tr><tr><td>Injekt-F single-use syringe</td><td>Braun</td><td>91 66 017V</td><td></td></tr><tr><td>Keck clips</td><td>Schott</td><td>KC29</td><td></td></tr><tr><td>L-&amp;alpha;-Phosphatidylcholine, 20% (Soy)</td><td>Avanti Polar Lipids</td><td>5416016</td><td>store under inert gas at -20 &amp;deg;C</td></tr><tr><td>NaCl 99.5% p.a.</td><td>Roth</td><td>3957.2</td><td></td></tr><tr><td>Nanopore E100 wafer/chips</td><td>Micromotive (Mainz/Germany)</td><td></td><td>available on request</td></tr><tr><td>Nucleopore Track-Etch Membrane 0.4 &amp;micro;m</td><td>Whatman</td><td>800282</td><td></td></tr><tr><td>Oregon Green Dextran 488 (70 kDa)</td><td>life Technologies</td><td>D-7173</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Oy647</td><td>Luminartis (M&amp;uuml;nster/Germany)</td><td>OY-647-T-1mg</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Rotilabo-syringe filtration, unsterile, pore-size 0.22 &amp;micro;m</td><td>Roth</td><td>P 818.1</td><td></td></tr><tr><td>Sephadex G-50</td><td>Sigma-Aldrich</td><td>G5080</td><td>column material for size exclusion chromatography</td></tr><tr><td>Silastic MDX4-4210</td><td>Dow Corning</td><td></td><td>curing agent for chip fixation onto cover glass support</td></tr><tr><td>sticky-Slide 8-well</td><td>ibidi</td><td>80828</td><td>multi-well chamber for the mounting onto glass slides (chip holder)</td></tr><tr><td>Three-way stopcock blue</td><td>Sarstedt</td><td>744410001</td><td></td></tr><tr><td>Tris Pufferan 99.9% Ultra Quality</td><td>Roth</td><td>5429.2</td><td></td></tr><tr><td>Triton-X 100</td><td>Roth</td><td>6683.1</td><td></td></tr><tr><td>Whatman 0.2 &amp;micro;m cellulose nitrate membrane filter</td><td>Roth</td><td>NH69.1</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B&amp;uuml;chi 461 water bath</td><td>B&amp;uuml;chi</td><td></td><td></td></tr><tr><td>B&amp;uuml;chi Rotavapor RE 111</td><td>B&amp;uuml;chi</td><td></td><td></td></tr><tr><td>Cary Eclipse Fluorescence Spectrophtometer&amp;nbsp;</td><td>Varian</td><td></td><td></td></tr><tr><td>LiposoFast Mini Extruder</td><td>Avestin</td><td></td><td></td></tr><tr><td>Membrane pump&amp;nbsp;</td><td>Vaccubrand</td><td>15430</td><td></td></tr><tr><td>Nanosight Nanoparticle Tracking Microscope</td><td>Malvern / Nanosight</td><td>LM 14C</td><td></td></tr><tr><td>NyONE microscope</td><td>Synentec</td><td></td><td>available on request</td></tr><tr><td>Pump control</td><td>Vaccubrand</td><td>CVC 2II</td><td></td></tr><tr><td>Sonicator bath Sonorex RK100H</td><td>Brandelin electronic</td><td>31200001107477</td><td></td></tr><tr><td>Vaccum pump RC5</td><td>Vaccubrand</td><td>1805400204</td><td></td></tr><tr><td>Water bath W13</td><td>Haake</td><td>002-9910</td><td></td></tr><tr><td>Plasma Cleaner PDC-37G</td><td>Harrick Plasma</td><td>PDC-37G</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td>Open Source</td><td>http://imagej.nih.gov/ij/</td><td>scientific image processing software</td></tr><tr><td>NanoCalcFX</td><td>Freeware</td><td>http://sourceforge.net/projects/nanocalc/</td><td>data analysis/evaluation software for massive transport kinetic datasets</td></tr><tr><td>NTA 2.3 Analytical Software</td><td>Nanosight</td><td></td><td>data acquisition and analysis software for nanoparticle tracking microscope</td></tr><tr><td>NTA 2.3 Temperature Comms</td><td>Nanosight</td><td></td><td>temperature controle software for nanoparticle tracking microscope</td></tr></tbody></table>

membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution

transloke substrat olmayan electrogenic ise tek bir protein düzeyinde membran proteini taşıma hala detaylı analizler kaçıyor. Kaydadeğer çaba bu alanda yapılmış, ancak zar taşıyıcıları analizi için gerekli olan solvent içermeyen çift katlı lipid teknikleri ile birlikte otomatik yüksek verimli taşıma analizi sağlayan teknikler nadirdir. taşıyıcıların Bu sınıf, ancak hücre homeostazında önemli ve bu nedenle ilaç geliştirme ve yeni anlayışlar kazanmak için metodolojiler önemli bir hedef umutsuzca ihtiyaç olduğunu. Burada sunulan el yazması tek taşıyıcı çözünürlükte membran proteini aracılı taşıma süreçlerinin analizi için yeni bir Biochip kurulmasını ve kullanımını açıklar. biyoçip tasarımında oldukça paraleldir ve endüstriyel cins ve miktarda üretilebilir nanopores çevrelediği, mikro boşlukların üst oluşmaktadır. Protein Barınma lipozomlar doğrudan uygulanabilirSSM-teknikleri kullanarak kendini monte gözenek kapsayan çift katlı lipit katmanını oluşturan yonga yüzeyi (katı lipid membranlar desteklenir). membran parçaları, müstakil içine veya gerçek zamanlı multi-spektral floresan okuma ile takip edilebilir kavite alanı, dışarı substrat translokasyonu için arayüz sağlıyor Gözenek kapsayan. standart operasyon prosedürleri (SOP) kurulması işlevsel yeniden olabilir hemen hemen her membran proteini çip yüzeyinde protein barındıran lipit tabakalarının kolay kurulmasını sağlar. tek ön şart olmayan electrogenic taşıma yüzeyler için bir floresan okuma-out sistemi kurulmasıdır. Yüksek içerik tarama uygulamaları paralel olarak birden fazla fiş kayıt otomatik ters floresan mikroskop kullanılarak accomplishable vardır. Büyük veri kümeleri serbestçe kullanılabilir özel tasarlanmış bilgisayar programları kullanılarak analiz edilebilir. Üç renkli çoklu spektral floresanOkuması ayrıca yanlış pozitif sonuçlar ortadan kaldırarak, farklı olay sınıflarına tarafsız veri ayrımcılık sağlar. Chip teknolojisi şu anda SiO 2 yüzeylere göre, ama altın kaplamalı yonga yüzeyleri kullanılarak daha fonksiyonlandırmalar da mümkündür edilir.

Gözenek kapsayan zarlar kolayca kendi kendine monte şekilde nano yapılı çip yüzeyinde oluşturulabilir. Ancak altta yatan süreç hala hassas ve lipozom boyutu, lipozom nüfus, lameller, lipid-ve tuz konsantrasyonu ve kimyasal yüzey özelliklerinin monodispersiteye gibi birçok parametrede tarafından etkilenir. Bu parametrelerin çoğu dikkatle, özelliği yukarıdaki protokol standardize edilmiştir. Ancak her yeni hazırlanması sırasında kontrol edilmelidir Diğer parametrel...

Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membran Transport Tek Protein çözünürlükte Oldukça Paralel nanopore çip Sistemi Analiz Süreçleri

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

membrane-transport-processes-analyzed-highly-parallel-nanopore-chip

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.7K Görüntüleme.  Goethe University Frankfurt. The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Video: Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

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

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

Boyutu ince ayar ve Solid-state nanopores Gürültü en aza indirmek

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the ...

Mühendislik

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state Nanopores ile izlenmesi Protein Adsorpsiyon

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Biyomühendislik

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Nanopore sıralamayı kullanarak tüm kan mRNA sıralaması

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Genetik

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton pompalama membran enzimler elektrokimya ve floresan mikroskopisi kullanılarak incelenmesi için tek lipozom ölçümleri

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Tek metalik nanopartiküller yüksek çözünürlüklü fiziksel karakterizasyonu

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

Kimya

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Membran Protein-Protein Etkileşim Analizi için Yerli Hücreli Membran Nanopartiküller Sistemi

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

Tek nano tanecikleri analiz etmek için kapalı tip kablosuz Nanopore elektrot

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Membranla İlişkili Makromolekül Dinamiğini Karakterize Etmek için Kütleye Duyarlı Partikül İzleme

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...