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.

<ol>
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The authors declare no competing financial interests.

Die hier vorgestellte Technik ermöglicht eine hochparallele Analyse von Membranproteintransport. Rekonstituierten Membranprotein-Systeme können direkt auf dem Biochip aufgebracht werden, so dass die Anpassung der theoretisch jeder Membrantransporter oder möglichen Kanal. Transport-Analyse wird durch die Einrichtung eines fluoreszierenden Auslesesystem, entweder durch direkte Fluoreszenzänderung (Translokation von Fluorophore oder fluoreszenzmarkierte Substrate) oder indirekte Fluoreszenzänderung (pH-sensitiven Farb...

Die Analyse von Membranproteinen ist von Interesse in den letzten 20 Jahren für die Grund- und pharmazeutischen Forschung erhöht werden. Die Entwicklung neuer Medikamente ist abhängig von der Identifizierung und detaillierte Charakterisierung neuer Ziele, die derzeit einer der limitierenden Faktoren zu sein. Die Tatsache , dass etwa 60% aller drug targets sind Membranproteine ​​1, macht die Entwicklung von Techniken , um ihre Funktion wichtigsten aufzuklären. 4 - In der Vergangenheit wurden in 2 Vielzahl entwickelten Techniken für die Untersuchung von elektrogenen Kanäle und Transporter. Nicht elektrogener Substrate im Gegensatz eine schwierige Aufgabe darstellen. Sie sind jedoch von besonderem Interesse als Hauptangriffspunkte für Medikamente, da sie den Fluss von gelösten Stoffen und Nährstoffen durch die Zellmembran und die Funktion als Schlüsselrezeptoren in Signalkaskaden 5 steuern. Beträchtliche Anstrengungen wurden in die Entwicklung von t gesetztechniques die Funktion von Membrantransportproteinen 6, 7 zu untersuchen. 10, darunter auch feste unterstützten Lipid - Doppelschichten, tethered Bilayer 11, 12, microblack Lipidmembranen 13, 14 und nativen Vesikel - Arrays 15, 16 ein paar zu nennen - Systeme festen Träger unter Verwendung von Membranen wurden als vielversprechendsten Instrumente auf diesem Gebiet 8 entstanden. Einige von ihnen sind sogar als Handelsaufbauten 17, 18. Einige Beispiele wurden veröffentlicht Kombination die Fähigkeit einzelne Membranproteine ​​in einer hochparallelen Weise 14 zu studieren, 19, Voraussetzung für Screening - Anwendungen. Allerdings überbrücken diese Methoden nur selten aus der Grundlagenforschung zur industriellen Umfeld. Die Schwierigkeiten liegen oft in der Fähigkeit des Systems, automatisierbar zu sein, die kostenintensive Herstellung und / oder aufwendige Vorbereitung. Ein Ansatz overcoming alle oben erwähnten Hindernisse ist das Endziel. Die Technik , die hier vorgestellt wurde entwickelt Membrankanäle und Transporter in vitro in einer kontrollierten Umgebung auf dem einzelnen Protein - Ebene 20 zu studieren - 22. Die Rekonstitution von gereinigten Membranproteinen in LUVs ist viel mehr etabliert als vergleichbare Ansätze für GUVs 23-26 oder schwarzen Lipidmembranen 27. Sie können direkt auf die Chipoberfläche aufgebracht werden, in dem Doppelschichtbildung erfolgt über eine Selbstorganisationsprozess stattfindet. Der Glasboden Design des nanoporösen Chip (Fig. 1) ermöglicht eine Luft Mikroskopie, die die einfache Automatisierung des Systems ermöglicht. In Kombination mit einer motorisierten Stufe kann mehrere Chips gleichzeitig gemessen werden, wobei jedes Sichtfeld Tausende von abgedichteten Hohlräumen zur Analyse enthält. Abbildung 1. Design von gemultiplexten Nanopore Biochips. A) A silicon-on-insulator (SOI) Wafers durch reaktives Ionenätzen strukturiert. Ungefähr 1.150 einzelne Chips werden von jedem Wafer hergestellt mit identischen Eigenschaften und Qualität. B) Jeder Chip umfasst 250.000 einzelne Mikrokavitäten mit Nano - Öffnungen. Maßstabsbalken:. 200 &amp; mgr; m C) Jeder Hohlraum ist adressierbar über multispektrale Fluoreszenz ausgelesen. Eine undurchsichtige Deckschicht blockiert die Fluoreszenzsignale aus dem Pufferspeicher, so dass der Biochip kompatibel mit invertierten Fluoreszenzmikroskop. D) Rasterkraftmikroskopie (AFM) Bildgebung Porenöffnungen und Oberflächenrauhigkeit der Siliziumdioxid - Schicht von 3,6 nm gleichmäßig angeordnet enthüllt (n = 40) optimal für Vesikelfusion. Maßstabsbalken:. 5 um E) Rasterelektronen microscopy (SEM) Aufnahme zeigt einen Querschnitt durch die Nanopore die den Zugang zu den Femtoliter Hohlräume innerhalb des Siliziumchips. Diese Zahl wurde mit Erlaubnis wiederverwendet werden von 21. <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Alle Datenanalyse wird mit Freeware durchgeführt uneingeschränkten Zugriff für Endanwender zu gewährleisten. Zeitreihen sind mit dem kostenlosen Bildverarbeitungssoftware analysiert und eine benutzerdefinierte Build-Kurvenanalyse-Software ermöglicht die Stapelverarbeitung und einfache Korrelation von großen Datensätzen mit mehreren Fluoreszenzkanälen und Tausende von Kurven. Das Modellprotein in diesem Protokoll wird die mechanosensitive Kanal von großer Leitfähigkeit (MscL) Kanalproteins von E. abgeleitet coli. Es fungiert als ein Ventil in der Natur osmotischen Schock freizusetzen, wurde aber derart modifiziert, dass synt rational entworfenhetic Funktionalitäten können kovalent an die Kanäle Einschnürung Seite angebracht werden. Via Ladungs ​​Abstoßung des kovalent gebundenen Aktivator (MTSET) der Kanal ausgelöst wird, zu öffnen, die Schaffung eines Nano-Ventil. Kleine Moleküle, wie Ionen, Wasser, kleine Proteine, aber auch kleine Fluorophore können durch den Kanal zu durchdringen. Hierbei wird das Protein als ein Modell verwendet, um die Fähigkeit des Systems zu demonstrieren Protein-vermittelte Translokation zu detektieren.

<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

Membranproteintransport auf dem einzelnen Proteinebene ausweicht noch detaillierte Analyse, wenn das transloziert Substrat nicht elektrogener ist. Erhebliche Anstrengungen wurden in diesem Bereich, sondern Techniken ermöglicht automatisierte Hochdurchsatz-Transport-Analyse in Kombination mit lösemittelfreien Techniken Lipid-Doppelschicht für die Analyse von Membrantransportern sind erforderlich selten. Diese Klasse von Transportern ist jedoch von entscheidender Bedeutung in der Zelle Homöostase und daher ein zentrales Ziel in der Medikamentenentwicklung und Methoden neue Erkenntnisse dringend benötigt zu gewinnen. Die hier präsentierten Handschrift beschreibt den Aufbau und die Handhabung eines neuartigen Biochips für die Analyse von Membranprotein-vermittelte Transportprozesse auf Einzel transporter Auflösung. Der Biochip besteht aus Mikrokavitäten durch Nanoporen eingeschlossen, die stark parallel in seiner Gestaltung und kann in Industriequalität und Menge hergestellt werden. Protein-tragende Liposomen können direkt angewendet werdendie Chipoberfläche bilden selbstorganisierende poren Spanning Lipid-Doppelschichten SSM-Techniken (feste Lipidmembranen unterstützt). Pore-Spanning Teile der Membran sind freistehend, Bereitstellen der Schnittstelle für das Substrat Translokation in den oder aus dem Hohlraum, die durch multispektrale Fluoreszenzanzeige in Echtzeit verfolgt werden können. Die Einrichtung von Standard Operating Procedures (SOPs) ermöglicht die einfache Einrichtung von Protein-tragenden Lipid-Doppelschichten auf der Chipoberfläche praktisch jeder Membranprotein, das funktionell wiederhergestellt werden kann. Einzige Voraussetzung ist die Schaffung eines Fluoreszenzlesesystem für Nicht-elektrogener Transportgründen. High-Content-Screening-Anwendungen sind erfüllbar durch den Einsatz von automatisierten inversen Fluoreszenzmikroskope mehrere Chips parallel aufzuzeichnen. Große Datenmengen können mit dem frei verfügbaren anwendungsspezifische Analyse-Software analysiert werden. Drei-Farben-Mehrspektrale FluoreszenzAuslesen erlaubt darüber hinaus für unvoreingenommene Daten Diskriminierung in verschiedenen Ereignisklassen, falsch positive Ergebnisse zu beseitigen. Die Chip - Technologie basiert derzeit auf SiO 2 -Oberflächen, aber weitere Funktionalisierung goldbeschichteten Spanflächen ist ebenfalls möglich.

Pore-Spanning Membranen können leicht auf der Oberfläche nanostrukturierten Chip in einer selbstorganisierenden Weise erstellt werden. Doch der zugrunde liegende Prozess ist noch zart und von vielen Parametern wie Liposomgröße, Monodispersität der Liposomen-Population, Lamellarität, Lipid- und Salz-Konzentration und chemischen Oberflächeneigenschaften beeinflusst. Die meisten dieser Parameter sind in dem obigen Protokoll sorgfältig charakterisiert und standardisiert. Andere Param...

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.

Membrantransportprozesse durch eine hochparallele Nanopore Chip-System bei Einzel Protein Resolution Analysierte

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

Research

JoVE Journal

Biology

11.6K Aufrufe.  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.

Serie: 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.

Verfahren zum Visualisieren und Analysieren Membran interagierender Proteine ​​durch Transmissionselektronenmikroskopie

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

Forschung

Biochemie

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.

Feinabstimmung der Größe und Minimierung der Lärm von Solid-State-Nanoporen

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

Ingenieurwesen

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.

Überwachung Proteinadsorption mit Solid-State-Nanoporen

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

Biotechnik

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.

Einzelne Liposomen Messungen für das Studium der Protonen-Pumpen Membran Enzyme mit Elektrochemie und Fluoreszenz-Mikroskopie

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.

Physikalische Charakterisierung einzelner metallischer Nanopartikel in hoher Auflösung

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

Chemie

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.

Native Cell Membrane Nanoparticles System für Membran-Protein-Protein-Interaktionsanalyse

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.

Eine geschlossene drahtlose Nanopore Elektrode für die Analyse von einzelnen Nanopartikel

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

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Messung der Nukleotidbindung an intakte, funktionelle Membranproteine in Echtzeit

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

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.

Massensensitive Partikelverfolgung zur Charakterisierung der membranassoziierten Makromoleküldynamik

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

Membrantransportprozesse durch eine hochparallele Nanopore Chip-System bei Einzel Protein Resolution Analysierte

Zusammenfassung

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Membrantransportprozesse durch eine hochparallele Nanopore Chip-System bei Einzel Protein Resolution Analysierte

Zusammenfassung

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