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.

여기에 제시된 기술은 막 단백질 수송의 고도의 병렬 분석을 할 수 있습니다. 재구성 세포막 단백질 시스템은 직접 이론적마다 막 수송 체의 적응을 가능 또는 채널은 바이오 칩에 적용될 수있다. 전송 분석은 직접 형광 변화 (형광의 전위 또는 형광 표지 된 기판) 또는 간접 형광 변화 (pH 민감성 염료, 보조 효소 반응)을 통해 중, 형광 판독 시스템의 구축에 의해 제한된다. 후자는 그러나 아직 확?...

막 단백질의 분석은 지난 20 년 동안 기본 및 제약 연구에 대한 관심이 증가되고있다. 신약 개발은 현재 제한 요인 중 하나 인, 식별 및 새로운 목표의 상세한 특성에 의존한다. 모든 약물 표적의 약 60 %는 단백질 막 한 사실이 기술의 개발은 그 기능이 가장 중요 규명 할 수있다. 4 - 과거 electrogenic 채널 및 수송을 연구하는 기술이 다수 개발되었다. 반면에 비 electrogenic 기판은 더 어려운 과제를 제시한다. 그들은 캐스케이드 5 시그널링 키 수용체로서 세포막 기능 및 전체 용질 및 영양소의 유동을 제어하는대로 그러나 프라임 약물 표적으로서 특별한 관심이다. 상당한 노력이 t의 개발에 투입하고있다echniques은 막 수송 단백질 (6), (7)의 기능을 연구한다. 몇 가지 이름 고체 지원 지질 이중층, 닿는 이중층 11, 12, microblack 지질 막 13, 14 및 기본 소포 어레이 (15, 16)을 포함하여 10 일 - 고체 지원 멤브레인을 사용하는 시스템은이 분야 8로 가장 유망한 도구가 등장했다. 그들 중 일부는 상업 설정 17, 18으로도 사용할 수 있습니다. 몇 가지 예는 고도의 병렬 14, 19, 심사 애플리케이션을위한 전제 조건에서 하나의 막 단백질을 연구 할 수있는 능력을 결합 발표되었다. 그러나 이러한 방법은 거의 산업 환경에 대한 기초 연구에서 다리 없다. 어려움은 종종 자동화가 될 수있는 시스템의 능력, 비용 집약적 생산 및 / 또는 준비에 수고 거짓말. 접근 오위에서 언급 한 모든 장애물을 vercoming하는 최종 목표이다. 22 - 여기에 제시된 기술은 단일 단백질 수준 (20)에 제어 된 환경에서 시험 관내에서 막 채널 수송을 연구하기 위해 개발되었다. 26 검은 지질 막 (27) - LUVs로 정제 막 단백질의 재구성 훨씬 더 GUVs (23)에 대한 비교 방법보다 설정됩니다. 이들은 직접 이중층의 형성이 자기 ​​조립 과정을 통해 이루어지고, 칩 표면에 적용될 수있다. 나노 다공성 칩 (도. 1)의 유리 바닥 디자인은 시스템의 간단한 자동화 허용 공기 현미경을 허용한다. 전동 스테이지와 함께 다중 칩 분석 밀봉 공동 수천 함유 뷰의 각 필드와 동시에 측정 될 수있다. 그림 1. 다중화 된 나노 기공 바이오칩의 설계. A)는 실리콘 - 온 - 인슐레이터 (SOI) 웨이퍼에 반응성 이온 에칭에 의해 구성된다. 약 1,150 개별 칩은 동일한 특성 및 품질. B) 각각의 칩은 나노 구멍에 25 ​​만 개별 마이크로 공동을 포함하는 각각의 웨이퍼에서 제조된다. 스케일 바 :. 200 μm의 C) 각 공동 멀티 스펙트럼 형광 판독을 통해 주소입니다. 반전 된 형광 현미경. D) 원 자간 력 현미경으로 바이오칩 호환 만들기는 불투명 한 상부층 블록 버퍼 탱크에서 형광 신호 (AFM) 촬상 균등 (세공 개구 3.6 nm 인 이산화 규소 층의 표면 거칠기를 배치 계시 N = 소포 융합 40) 최적. 스케일 바 :. 5 μm의 E) 주사 전자 마이크roscopy (SEM) 이미지는 실리콘 칩 내부 femtoliter 캐비티에 대한 액세스를 허용 나노 기공을 통한 단면을 도시한다. 이 그림은 (21)로부터 허가를 재사용했다. <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 모든 데이터 분석은 최종 사용자가 제한되지 않은 액세스를 실현 프리웨어를 사용하여 수행된다. 시계열없는 화상 처리 소프트웨어 맞춤 제작 곡선 분석 소프트웨어 있도록 배치 프로세싱 곡선 여러 형광 채널 수천 대용량 데이터의 직접적인 상관 관계를 이용하여 분석 하였다. 이 프로토콜에서 사용되는 모델 단백질은 E. 유래의 컨덕턴스 (후 MsCl) 채널 단백질의 채널이다 mechanosensitive 대장균. 그것은 본질적으로 삼투압 충격을 해제하기위한 밸브로서 기능하지만 SYNT 합리적으로 설계하는 방식으로 수정hetic 기능은 공유 채널 수축 측에 부착 될 수있다. 나노 밸브를 만드는 채널이 열 트리거되는 공유 결합 활성제 (MTSET)의 전하 반발, 비아. 이온, 물, 작은 단백질뿐만 아니라, 작은 형광체 같은 작은 분자는 채널을 통해 투과 할 수있다. 여기서, 단백질은 단백질 - 매개 전위를 검출하기위한 시스템의 능력을 증명하기위한 모델로서 사용된다.

<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

전좌 기판 비 electrogenic 경우 단일 단백질 수준에서 세포막 단백질 수송 여전히 상세한 분석을 기피한다. 상당한 노력이 분야에서 제조되었지만, 막 수송의 분석에 필요한 무용제 지질 이중층 기술과 조합하여 높은 처리량 자동 반송 분석을 가능 기술들은 드물다. 운송의이 클래스는 그러나 세포 항상성에 중요한 따라서 신약 개발과 새로운 통찰력을 얻을 수있는 방법론의 주요 대상이 절실히 필요하다. 여기에 제시된 논문은 단일 트랜스 해상도 세포막 단백질 매개 수송 과정의 분석을위한 신규 한 바이오칩의 확립 및 처리를 설명. 바이오칩은 설계에서 매우 평행하고 산업 등급과 양으로 제조 할 수 나노 기공으로 둘러싸인 미세 공동으로 구성된다. 단백질 형질 리포좀에 직접 적용 할 수있다SSM-기술을 이용하여 자기 조립 기공 걸친 지질 이중층을 구성하는 칩면 (고체 지질 막을 지원됨). 막의 일부가 자립으로 또는 실시간 다중 분광 형광 판독 하였다 가능 캐비티 공간에서 기판 전위에 대한 인터페이스를 제공하는 세공은 스패닝. 표준 작업 절차 (SOP)의 설립은 기능적으로 재구성 될 수있는 거의 모든 막 단백질 칩 표면에 단백질 형질 지질 이중층의 간단한 구축을 허용한다. 유일한 필수 비 electrogenic 기판 반송 용 형광 판독 시스템의 구축이다. 하이 콘텐츠 스크리닝 응용 병렬 다중 칩 녹화 자동 반전 된 형광 현미경을 사용하여 accomplishable이다. 큰 데이터 세트는 자유롭게 사용할 맞춤 설계 해석 소프트웨어를 사용하여 분석 될 수있다. 세 가지 색 멀티 스펙트럼 형광판독 또한 거짓 긍정적 인 결과를 제거, 다른 이벤트 클래스로 편견 데이터 차별을 허용합니다. 칩 기술은 현재의 SiO2 표면을 기반으로하지만, 금 코팅 칩 표면을 사용하여 추가로 작용하는 것도 가능하다.

기공 멤브레인 스패닝 쉽게 자기 조립 방식으로 나노 칩 표면 상에 생성 될 수있다. 그러나 기본 프로세스는 여전히 섬세하고 리포좀의 크기, 리포좀 인구, lamellarity, 지질 및 소금 농도와 화학적 표면 특성의 단 분산 같은 많은 매개 변수에 의해 영향을 받는다. 이러한 매개 변수의 대부분은주의 깊게 특징 및 위의 프로토콜 표준화되었다. 그러나 모든 새로운 제조시 확인?...

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.

멤브레인 운송은 단일 단백질 해상도에서 고도의 병렬 나노 기공 칩 시스템에 의해 분석 처리합니다

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 조회수.  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.

비디오: 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.

방법은 시각화 및 투과 전자 현미경에 의해 막 상호 작용하는 단백질을 분석하는

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

연구

생화학

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.

크기를 미세 조정 및 고체 나노 세공의 잡음을 최소화

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

공학

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.

고체 Nanopores와 모니터링 단백질 흡착

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

생체공학

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.

전기 화학 및 형광 현미경 검사 법을 사용 하 여 양성자 펌프 막 효소의 연구에 대 한 단일 Liposome 측정

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.

단일 금속 나노 입자의 고분해능 물리적 특성화

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

화학

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.

멤브레인 단백질-단백질 상호 작용 분석을 위한 기본 세포막 나노입자 시스템

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.

단일 나노 입자 분석에 대 한 폐쇄-타입 무선 Nanopore 전극

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.

온전한 기능성 막 단백질에 대한 뉴클레오티드 결합을 실시간으로 측정

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.

막과 관련된 거대분자 역학을 특성화하기 위한 질량 민감성 입자 추적

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