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.

ここで紹介する手法は、膜タンパク質輸送の高度な並列分析を可能にします。再構成された膜タンパク質系は、直接、理論的にすべての膜輸送体またはチャネル可能の適応を行う、バイオチップに適用することができます。輸送解析は、唯一の直接蛍光変化（蛍光体の転座または蛍光標識された基質）または間接的な蛍光変化（pH感受性色素、二次酵素反応）のいずれかを介して、蛍光読み?...

膜タンパク質の分析は、過去20年間で基礎および医薬品の研究のための関心が高まっになっています。新規薬剤の開発は、現在、制限要因の一つである、新たな標的の同定および詳細な特性に依存します。すべての薬物標的の約60％が膜タンパク質1であるという事実は、最も重要なその機能を解明するための技術の開発を行います。 4 -過去には、起電チャンネルおよびトランスポーターの研究のための技術は、多数の2で開発されてきました。逆における非起電性基板は、より多くの困難な課題を提示します。彼らはカスケード5のシグナル伝達に重要な受容体などの細胞膜と機能を横切る溶質および栄養素のフラックスを制御するように、彼らは、しかしプライム薬物標的としての特別な関心のです。 かなりの努力がトンの開発に置かれていますechniques膜輸送タンパク質6、7の機能を研究します。いくつか例を挙げると固体担持脂質二重層、繋留二重層11、12、microblackの脂質膜13、14およびネイティブベシクルアレイ15、16を含む10、 -固体支持膜を用いたシステムでは、このフィールド8として最も有望なツールを浮上しています。それらのいくつかは、市販のセットアップ17、18のようにしてもご利用いただけます。いくつかの実施例は、高度に並列に14、19、スクリーニング用途のための前提条件で単一の膜タンパク質を研究する能力を組み合わせること公開されています。しかし、これらの方法はほとんどの産業環境への基礎研究から橋渡しません。困難は、多くの場合、自動化するシステムの能力は、コストのかかる生産および/または面倒な準備にあります。アプローチoを上記の全ての障害をvercomingすることは、最終的な目標です。 22 -ここで紹介する手法は、単一のタンパク質レベル20に制御された環境で、in vitroで膜チャネルおよびトランスポーターを研究するために開発されました。 26または黒色脂質膜27 - LUVをに精製された膜タンパク質の再構成は、はるかにGUVs 23で同等のアプローチよりも確立されています。二重層の形成は自己集合プロセスを介して行われている場合、それらは直接チップ表面に適用することができます。ナノ多孔性チップ（ 図 1）のガラスボトム設計により、システムの簡単な自動化を可能にする空気顕微鏡を可能にします。電動ステージとの組み合わせで複数のチップを、分析のために密封された空洞の数千人を含むビューの各フィールドで、同時に測定することができます。 図 1。多重化されたナノ細孔のバイオチップの設計 A）は、シリコン・オン・インシュレータ（SOI）ウエハを反応性イオンエッチングにより構成されています。約1150個々のチップは、同一の特性と品質。B）各チップは、ナノ開口を備え25万個々のマイクロキャビティを備えるとともに、各ウェハから製造されます。スケールバー：200μmのC）各キャビティは、マルチスペクトル蛍光読み出しを経由してアドレス可能です。 intransparent上層倒立蛍光顕微鏡とバイオチップの互換性行うブロックバッファリザーバからの蛍光シグナルを、D）原子間力顕微鏡（AFM）イメージングが均一に配置された孔の開口部および3.6ナノメートルの二酸化ケイ素層の表面粗さを（明らかに、N = 40）小胞の融合のために最適。スケールバー：5μmのE）走査型電子マイクroscopy（SEM）画像は、シリコンチップ内のフェムトリットルの空洞へのアクセスを可能にするナノ細孔の断面を示しています。この数字は21から許可を得て再利用した。 <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> すべてのデータ分析は、エンドユーザのための無制限のアクセスを保証するためにフリーウェアを使用して実行されます。時系列は、フリーの画像処理ソフトウェアとバッチ処理と曲線の複数の蛍光チャネルおよび何千も大きなデータセットの単純な相関関係を可能にするカスタムビルド曲線分析ソフトウェアを用いて分析します。 このプロトコルで使用されるモデルタンパク質は、E由来大コンダクタンス（MSCL）チャネルタンパク質の機械受容チャネルであります大腸菌 。それは、本質的に浸透圧ショックを放出する弁として機能するが、合理的にSYNT設計ように変更されましたhetic機能は、共有チャネルの収縮側に取り付けることができます。共有結合した活性化剤（MTSET）の電荷反発を経由してチャンネルがナノバルブを作成、開くようにトリガされます。イオン、水、小さなタンパク質だけでなく、小型の蛍光団のような小分子は、チャネルを介して透過することができます。ここでは、タンパク質は、タンパク質媒介転座を検出するシステムの能力を実証するためのモデルとして使用されます。

<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

転位した基板は、非起電性である場合、単一のタンパク質レベルでの膜タンパク質の輸送はまだ、詳細な分析を忌避します。かなりの努力がこの分野で行われているが、膜トランスポーターの解析に必要な溶媒を含まない脂質二重層の技術と組み合わせて、自動化ハイスループットトランスポート分析を可能にする技術はまれです。トランスポーターのこのクラスは、しかし、細胞の恒常性において非常に重要なので、薬剤開発や新たな洞察を得るための方法論における重要な目標は必死に必要です。 ここで紹介する原稿は、単一のトランスポーター解像度で膜タンパク質媒介輸送プロセスの分析のための新規バイオチップの確立と取り扱いについて説明します。バイオチップは、その設計において非常に平行であり、工業用グレードおよび量で製造することができるナノポアで囲まれたマイクロキャビティから構成されています。プロテイン保有するリポソームは、直接に適用することができますSSM-技術（固体担持脂質膜）を用いた自己組織化細孔スパニング脂質二重層を形成するチップ表面。膜のポア貫通部分は、中またはリアルタイムでマルチスペクトル蛍光読み出しを続けることができ、キャビティ空間のうち基板転位のためのインターフェースを提供し、自立しています。標準作業手順書（SOP）の設立は、機能的に再構成することができる事実上すべての膜タンパク質のチップ表面上のタンパク質-保有脂質二重層の簡単な設置を可能にします。唯一の前提条件は、非起電輸送基質用の蛍光読み出しシステムの確立で​​す。 ハイコンテンツスクリーニング用途は、並行して複数のチップを記録し、自動化倒立蛍光顕微鏡を使用することによって達成できるです。大きなデータセットを自由に利用できるカスタム設計解析ソフトウェアを用いて分析することができます。三色のマルチスペクトル蛍光読み出し、さらに、偽陽性の結果を排除し、別のイベントクラスに公平なデータ判別を可能にします。 チップ技術は、現在のSiO 2表面に基づいているが、金でコーティングされたチップ表面を使用してさらに官能化することも可能です。

ポア貫通膜は容易に自己集合したようにナノ構造チップ表面上に作成することができます。しかし、根本的なプロセスはまだ繊細でリポソームのサイズ、リポソーム集団の単分散性、ラメラ、脂質や塩分濃度と化学的表面特性のような多くのパラメータの影響を受けています。これらのパラメータのほとんどは、慎重に特徴付けられ、上記のプロトコルで標準化され?...

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.

単一のタンパク質解像度で高並列ナノ細孔チップシステムにより分析膜輸送プロセス

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 ビュー.  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.

ビデオ: 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.

サイズを微調整すると固体ナノ細孔のノイズを最小限

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

工学

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.

ソリッドステートナノ細孔を持つ監視タンパク質吸着

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

生物工学

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.

電気化学と蛍光顕微鏡を用いたプロトン ポンプ膜酵素の研究のための単一リポソーム測定

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

生化学

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.

単一金属ナノ粒子の高解像度物理特性評価

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

化学

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.

膜蛋白質相互作用解析用のネイティブ細胞膜ナノ粒子システム

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.

インタクトな機能的な膜タンパク質へのヌクレオチド結合をリアルタイムで測定

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>固体NMRによる天然膜タンパク質の高分解能研究

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.

固体NMRによる天然膜タンパク質の高分解能研究

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.

膜関連高分子ダイナミクスを特徴付けるための質量感受性粒子追跡

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.

核輸送の1分子イメージング

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

単一のタンパク質解像度で高並列ナノ細孔チップシステムにより分析膜輸送プロセス

要約

さらに動画を探す

この動画の章

サイズを微調整すると固体ナノ細孔のノイズを最小限

ソリッドステートナノ細孔を持つ監視タンパク質吸着

電気化学と蛍光顕微鏡を用いたプロトンポンプ膜酵素の研究のための単一リポソーム測定

単一金属ナノ粒子の高解像度物理特性評価

膜蛋白質相互作用解析用のネイティブ細胞膜ナノ粒子システム

インタクトな機能的な膜タンパク質へのヌクレオチド結合をリアルタイムで測定

固体NMRによる天然膜タンパク質の高分解能研究

固体NMRによる天然膜タンパク質の高分解能研究

膜関連高分子ダイナミクスを特徴付けるための質量感受性粒子追跡

核輸送の1分子イメージング

単一のタンパク質解像度で高並列ナノ細孔チップシステムにより分析膜輸送プロセス

要約

さらに動画を探す

この動画の章

サイズを微調整すると固体ナノ細孔のノイズを最小限

ソリッドステートナノ細孔を持つ監視タンパク質吸着

電気化学と蛍光顕微鏡を用いたプロトン ポンプ膜酵素の研究のための単一リポソーム測定

単一金属ナノ粒子の高解像度物理特性評価

膜蛋白質相互作用解析用のネイティブ細胞膜ナノ粒子システム

インタクトな機能的な膜タンパク質へのヌクレオチド結合をリアルタイムで測定

固体NMRによる天然膜タンパク質の高分解能研究

固体NMRによる天然膜タンパク質の高分解能研究

膜関連高分子ダイナミクスを特徴付けるための質量感受性粒子追跡

核輸送の1分子イメージング

電気化学と蛍光顕微鏡を用いたプロトンポンプ膜酵素の研究のための単一リポソーム測定