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.

La tecnica qui presentata permette un&#39;analisi altamente parallela di trasporto delle proteine ​​di membrana. sistemi proteici di membrana ricostituiti possono essere direttamente applicati al biochip, rendendo l&#39;adattamento teoricamente ogni trasportatore di membrana o canale possibile. Analisi dei trasporti è limitata solo dalla creazione di un sistema di read-out a fluorescenza, sia attraverso il passaggio diretto di fluorescenza (traslocazione di fluorofori o substrati fluorescente) o il cambiamento di f...

L&#39;analisi delle proteine ​​di membrana è diventato di crescente interesse per la ricerca di base e farmaceutica negli ultimi 20 anni. Lo sviluppo di nuovi farmaci dipende identificazione e caratterizzazione dettagliata di nuovi bersagli, attualmente essendo uno dei fattori limitanti. Il fatto che circa il 60% di tutti i bersagli farmacologici sono proteine ​​di membrana 1, rende lo sviluppo di tecniche per chiarire la loro funzione più importante. In passato, le tecniche per lo studio dei canali elettrogeniche e trasportatori sono stati sviluppati in moltitudine di 2 - 4. substrati non elettrogeniche in contrario presentano un compito più impegnativo. Sono comunque di particolare interesse come obiettivi primari droga, come controllano il flusso di soluti e nutrienti attraverso la membrana cellulare e la funzione come recettori chiave in cascate di segnalazione 5. considerevole sforzo è stato messo nello sviluppo di techniques per studiare la funzione di trasportatore di membrana 6, 7. I sistemi che utilizzano membrane solidi supportati sono emersi come strumenti più promettenti in questo campo 8 - 10, compreso solidi doppi strati lipidici supportati, bistrati frenati 11, 12, membrane lipidiche microblack 13, 14 e nativi array vescicola 15, 16 solo per citarne alcuni. Alcuni di loro sono anche disponibili come configurazioni commerciali 17, 18. Alcuni esempi sono stati pubblicati combinando la capacità di studiare singole proteine ​​di membrana in modo altamente paralleli 14, 19, un prerequisito per applicazioni di screening. Tuttavia, questi metodi raramente colmare dalla ricerca di base per l&#39;ambiente industriale. Le difficoltà spesso trovano nella capacità del sistema di essere automatizzabili, la produzione costose e / o preparazione laboriosa. Un approccio overcoming tutti gli ostacoli di cui sopra è l&#39;obiettivo finale. La tecnica qui presentata è stato sviluppato per studiare canali di membrana e trasportatori in vitro in un ambiente controllato al livello proteico singola 20 - 22. Ricostituzione di proteine ​​di membrana purificate in luvs è molto più stabilito di approcci comparabili per GUVs 23 - 26 o lipidi nero membrane 27. Essi possono essere applicati direttamente alla superficie del chip, dove la formazione doppio strato avviene attraverso un processo di auto-assemblaggio. Il design fondo di vetro del chip nanoporoso (Fig. 1) permette per microscopia dell&#39;aria, che permette l&#39;automazione semplice del sistema. In combinazione con uno stadio motorizzato chip multipli possono essere misurate allo stesso tempo, con ogni campo di vista contenente migliaia di cavità sigillate per l&#39;analisi. Figura 1. Progettazione di biochip nanopori multiplex. A) una Silicon-on-Insulator (SOI) wafer è strutturata da incisione reattiva ioni di litio. Circa 1.150 singoli chip sono fabbricati da ogni wafer con proprietà identiche e qualità. B) Ogni chip comprende 250.000 microcavità individuali con aperture nano. Barra di scala:. 200 micron C) Ogni cavità è indirizzabile tramite fluorescenza multispettrale read-out. Un blocchi strato superiore non trasparenti i segnali fluorescenti dal serbatoio tampone, rendendo il biochip compatibile con microscopi a fluorescenza invertiti. D) microscopia a forza atomica (AFM) di imaging rivela organizzati uniformemente aperture dei pori e la rugosità superficiale dello strato di biossido di silicio di 3,6 nm (n = 40) ottimale per la fusione delle vescicole. Barra di scala:. 5 micron E) mic elettronico a scansioneimmagine roscopy (SEM) mostra una sezione trasversale attraverso il nanoporo consentendo l&#39;accesso alla cavità femtoliter all&#39;interno del chip di silicio. Questo dato è stato riutilizzato con il permesso di 21. <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> Tutte le analisi dei dati viene effettuata utilizzando freeware per garantire l&#39;accesso senza restrizioni per gli utenti finali. Le serie temporali vengono analizzati utilizzando il software gratuito di elaborazione delle immagini e di una elaborazione batch curva di generazione personalizzata software di analisi che permette e la correlazione diretta di dati di grandi dimensioni con canali fluorescenti multiple e migliaia di curve. La proteina modello utilizzato in questo protocollo è il canale meccanosensibili di proteine ​​grande conduttanza (MscL) canale derivato da E. coli. Esso funziona come una valvola per rilasciare shock osmotico in natura, ma è stato modificato in modo tale che razionalmente progettato syntfunzionalità hetic possono covalentemente essere fissati al cielo canali costrizione. Via carica-repulsione dell&#39;attivatore legame covalente (MTSET) il canale viene attivato per aprire, la creazione di un nano-valvola. Piccole molecole come ioni, acqua, piccole proteine, ma anche piccoli fluorofori possono permeare attraverso il canale. Qui, la proteina viene utilizzato come modello per dimostrare la capacità del sistema di rilevare la traslocazione di proteine ​​mediata.

<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

trasporto proteina di membrana a livello della proteina singolo elude ancora un&#39;analisi dettagliata, se il substrato traslocato non è elettrogenico. Notevoli sforzi sono stati fatti in questo campo, ma le tecniche che permettono automatizzato di analisi trasporto ad alta produttività in combinazione con lipidi tecniche di doppio strato privi di solventi necessari per l&#39;analisi dei trasportatori di membrana sono rari. Questa classe di trasportatori, tuttavia, è cruciale nella omeostasi cellulare e quindi un obiettivo chiave nello sviluppo di farmaci e metodologie per acquisire nuove conoscenze disperatamente bisogno. Il manoscritto qui presentato descrive la creazione e la gestione di un romanzo biochip per l&#39;analisi di proteine ​​mediata processi di trasporto di membrana con risoluzione singolo trasportatore. Il biochip è composto da microcavità racchiuse da nanopori che è altamente parallelo nel suo design e possono essere prodotti in grado industriale e quantità. liposomi Protein-nutrirebbero possono essere direttamente applicati aila superficie del chip che formano doppi strati lipidici poro-spanning auto-assemblate utilizzando SSM-tecniche (solido supportato membrane lipidiche). Pore-attraversa parti della membrana sono indipendenti, fornendo l&#39;interfaccia per substrato traslocazione dentro o fuori lo spazio della cavità, che può essere seguita da lettura fluorescente multispettrale in tempo reale. La definizione di procedure operative standard (SOP) permette la creazione diretta di doppi strati lipidici proteine ​​ospitare sulla superficie del chip di proteine ​​virtualmente ogni membrana che può essere ricostituito in modo funzionale. L&#39;unico requisito è la creazione di un sistema di read-out fluorescente per substrati di trasporto non elettrogeniche. applicazioni ad alto contenuto di screening sono attuabile mediante l&#39;uso di microscopi a fluorescenza automatizzati invertiti registrazione chip multipli in parallelo. Grandi insiemi di dati possono essere analizzati utilizzando il software di analisi custom-designed liberamente disponibile. Tre colori multi spettrale fluorescenteread-out, inoltre, consente una discriminazione dati imparziale in diverse classi di eventi, eliminando falsi risultati positivi. La tecnologia dei chip si basa attualmente su SiO 2 superfici, ma un&#39;ulteriore funzionalizzazione con superfici di chip d&#39;oro rivestite è inoltre possibile.

membrane Pore-spanning possono essere facilmente creati sulla superficie del chip nanostrutturata in maniera auto-assemblati. Tuttavia il processo di fondo è ancora delicata e influenzato da molti parametri come la dimensione dei liposomi, monodispersity della popolazione liposomi, lamellarity, lipid- e le proprietà del sale di concentramento e di superficie chimica. La maggior parte di questi parametri sono stati accuratamente caratterizzato e standardizzato nel protocollo di cui sopr...

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.

Trasporto di membrana processi analizzati da un altamente parallelo sistema Nanopore Chip a risoluzione della proteina singolo

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

11.7K Views.  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

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.

Ottimizzazione delle dimensioni e minimizzando il rumore di stato solido Nanopori

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

Ricerca

Ingegneria

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.

Monitoraggio delle proteine ​​con a stato solido Nanopori

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

Bioingegneria

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.

Liposoma singole misurazioni per lo studio degli enzimi di membrana Proton-pompaggio usando microscopia fluorescente ed elettrochimica

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

Biochimica

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.

Caratterizzazione fisica ad alta risoluzione di nanoparticelle metalliche singole

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

Chimica

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.

Sistema di nanoparticelle a membrana cellulare nativa per l'analisi dell'interazione proteina-proteina di membrana

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

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.

Misurare il legame nucleotidico a proteine di membrana intatte e funzionali in tempo reale

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.

<meta charset="utf8"></head><body>Studi ad alta risoluzione di proteine in membrane naturali mediante NMR allo stato solido

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.

Studi ad alta risoluzione di proteine in membrane naturali mediante NMR allo stato solido

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.

Tracciamento delle particelle sensibili alla massa per caratterizzare la dinamica delle macromolecole associate alla membrana

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

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Singola molecola Imaging dei trasporti nucleari

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Trasporto di membrana processi analizzati da un altamente parallelo sistema Nanopore Chip a risoluzione della proteina singolo

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ottimizzazione delle dimensioni e minimizzando il rumore di stato solido Nanopori

Monitoraggio delle proteine con a stato solido Nanopori

Liposoma singole misurazioni per lo studio degli enzimi di membrana Proton-pompaggio usando microscopia fluorescente ed elettrochimica

Caratterizzazione fisica ad alta risoluzione di nanoparticelle metalliche singole

Sistema di nanoparticelle a membrana cellulare nativa per l'analisi dell'interazione proteina-proteina di membrana

Misurare il legame nucleotidico a proteine di membrana intatte e funzionali in tempo reale

Studi ad alta risoluzione di proteine in membrane naturali mediante NMR allo stato solido

Studi ad alta risoluzione di proteine in membrane naturali mediante NMR allo stato solido

Tracciamento delle particelle sensibili alla massa per caratterizzare la dinamica delle macromolecole associate alla membrana

Singola molecola Imaging dei trasporti nucleari

Trasporto di membrana processi analizzati da un altamente parallelo sistema Nanopore Chip a risoluzione della proteina singolo

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ottimizzazione delle dimensioni e minimizzando il rumore di stato solido Nanopori

Monitoraggio delle proteine ​​con a stato solido Nanopori

Liposoma singole misurazioni per lo studio degli enzimi di membrana Proton-pompaggio usando microscopia fluorescente ed elettrochimica

Caratterizzazione fisica ad alta risoluzione di nanoparticelle metalliche singole

Sistema di nanoparticelle a membrana cellulare nativa per l'analisi dell'interazione proteina-proteina di membrana

Misurare il legame nucleotidico a proteine di membrana intatte e funzionali in tempo reale

Studi ad alta risoluzione di proteine in membrane naturali mediante NMR allo stato solido

Studi ad alta risoluzione di proteine in membrane naturali mediante NMR allo stato solido

Tracciamento delle particelle sensibili alla massa per caratterizzare la dinamica delle macromolecole associate alla membrana

Singola molecola Imaging dei trasporti nucleari

Monitoraggio delle proteine con a stato solido Nanopori