저자는 회계 SMhardBi (프로젝트 번호 200021_150088/1)를 위한 스위스 국립 과학 재단을 인정 합니다. SANS 실험은 스위스 spallation 중성자 소스 SINQ, 폴 Scherrer Instute, Villigen, 스위스에서 수행 했다. 따뜻하게 저자 박사 요 아 킴 Kohlbrecher SAN 실험과 함께 그의 지도 감사합니다. 높은 자기장에서 복굴절 측정 설치 높은 분야 자석 실험실 HFML, Nijmegen, 네덜란드에서 기존 설치에서 영감을 했다. 우리는 브루노 Pfister 복굴절 설치의 전자 제품 개발에 그의 도움에 대 한, 1 월 Corsano와 다니엘 Kiechl, 레이저의 정밀 하 고 손쉬운 맞춤을 허용 하는 프레임 워크를 구축 및 지속적인 기술 지원에 대 한 박사 베른하르트 콜 러 감사 합니다.

<ol>
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저자는 공개 없다.

복굴절 측정 했다 SAN과 함께에서 사용 하는 방법의 세부 계정을 생성 높은 자석으로 응답 Ln3 + 어셈블리 Isabettini 외 에 인지질 킬레이트 화 하기 위한 방법을 평가 하 실험 23 제안 된 제조 프로토콜은 또한 더 긴 DPPC와 DPPE DTPA 인지질으로 구성 하는 어셈블리에 대 한 또는 그들의 bilayer에 화학적 조작된 스테로이드 유도체를 포함에 대 한 적용 됩니다. 1...

Bicelles는 수많은 지질 혼합물에서 얻은 디스크 모양의 polymolecular 어셈블리. 1 , 2 , 3 , 4 , 5 그들은 널리 NMR 분광학에 의해 생체 막의 구조 특성에 대 한 사용 됩니다. 6 , 그러나 7 , 최근 노력 하고자 가능한 응용 프로그램의 필드를 확장 합니다. 5 , 8 , 9 가장 공부 bicelle 시스템은 1, 2의 혼합물의 구성 되어-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 구성 어셈블리 및 1, 2의 평면 부분-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) 가장자리를 포함 하는 인지질. 1 , 2 , 3 인지질 bilayer 지시 자체 조립된 polymolecular 구조의 건축 구성의 분자 기하학. 4 , 5 DMPE-DTPA와 교체 DHPC 높은 자석으로 대답 하 고 조정할 수 있는 bicelle 시스템을 생성합니다. 10 , 11 DMPC/DMPE-DTPA/Ln3 + (어 금 니 비율 4:1:1) bicelles 연관 많은 더 많은 상자성 란타넘족 이온 (Ln3 +) 향상 된 자기 반응의 결과로 하는 bilayer의 표면에. 10 또한, 교체 수용 DHPC 분자 DMPE-DTPA/Ln3 + 수 희석 방지 bicelles의 형성 11
평면 polymolecular 어셈블리의 자석 alignability는 그들의 전반적인 자기 에너지에 의해 결정
<img alt="Equation 1" src="/files/ftp_upload/56812/56812eq1v2.jpg">(1)
B는 자기 필드 강도 <img alt="Equation 2" src="/files/ftp_upload/56812/56812eq2.jpg"> 자석 상수, n 집계 수와 <img alt="Equation 3" src="/files/ftp_upload/56812/56812eq3.jpg"> 는 bilayer를 구성 하는 지질 분자 diamagnetic 자화 율 이방성. 따라서, 자기장에 DMPC/DMPE-DTPA/Ln3 + bicelles의 응답은 그들의 크기 (집계 숫자 n)와 분자 diamagnetic 자화 율 이방성 Δχ에 의해 지어진 다. 후자는 쉽게 달성 chelated Ln3 +의 특성을 변경 하 여. 12 , 13 , 14 , 15 소개 콜레스테롤 (철-오) 또는 다른 스테로이드 유도체는 bilayer에 집계 숫자 n와 어셈블리의 자기 자화 율 Δχ 튜닝의 가능성을 제공 합니다. 11 , 16 , 17 , 18 , 19 주어진된 지질 구성에 대 한 큰 어셈블리 포함 더 많은 지질 E탄 창 (큰 집계 숫자 n)에 기여 가능 결과 더 alignable 종에. DMPC/DHPC bicelles의 크기 예를 들면, 전통적으로 구성 지질 비율이 나 총 농도의 최적화를 통해 제어 됩니다. 20 , 21 , 22 이 DMPC/DMPE-DTPA/Ln3 + bicelles에, 있지만 난방 제공 시 소포를 bicelle에서 그들의 열 가역 변환을 짓는 옵션 추가. 기계와 같은 멤브레인 필터를 통해 압출 수는 vesicles의 형성을 의미 합니다. 자석으로 alignable bicelles는 5 ° C에 냉각 시 재생성 하 고 그들의 크기 기 선구자에서 결정 됩니다. 11 여기, 우리가에 초점을 가진 DMPC/DMPE-DTPA/Tm3 + 기계 제조 절차의 잠재력 (어 금 니 비율 4:1:1) 또는 DMPC/철-오/DMPE-DTPA/Tm3 + (어 금 니 비율 16:4:5:5) 참조 시스템으로. 프로세스 작업 다른 Ln3 + 보다 Tm3 +비슷하게 작동 합니다. 이러한 기술에서 제공 하는 가능성의 넓은 범위는 그림 1 에서 강조 표시 하 고 광범위 하 게 다른 논의. 23
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56812/56812fig1.jpg"> 
그림 1: 가능한 제작 절차의 개요 개요. 공부 자석으로 alignable Ln3 + chelating polymolecular 어셈블리는 구성 중 DMPC/DMPE-DTPA/Tm3 + (어 금 니 비율 4:1:1) 또는 DMPC/철-오/DMPE-DTPA/Tm3 + (어 금 니 비율 16:4:5:5). 건조 지질 영화 7.4의 pH 값에 50 m m 인산 염 버퍼 화 이며 총 지질 농도 15 m m. 지질 영화의 효과적인 수 분 요구도 녹고 사이클 (피트) 또는 열 및 냉각 사이클을 동결 (H &#38; C). H &#38; C 사이클 마지막 동결 단계, 녹고 후 샘플을 다시 생성 하거나 압출 추가 없이 사용할 수 있다면 그들은 시간의 연장된 기간 동안 냉동 보관 하는 샘플을 다시 생성에 필요 하다. 이 단계는 Isabettini 외에 의해 광범위 하 게 논의 된다. 23 극대 alignable polymolecular 어셈블리 지질 구성에 따라 서로 다른 어셈블리 아키텍처를 제공 하는, 달성 된다. Bicelle 크기와 자석 alignability nanopore 멤브레인 필터를 통해 압출 (Ext)에 의해 가변 이다. 제시 정렬 요소 Af 800, 400, 200, 또는 100 통해 압출 DMPC/철-오/DMPE-DTPA/Tm3 + (어 금 니 비율 16:4:5:5) 샘플의 2D 작은 각 중성자 산란 (SANS) 패턴에서 계산 된 숨 구멍 nm. 측정 없이 더 자세히 여기에 적용 되지 것입니다 bicelle 정렬 측정의 보완 수단입니다. 11 , 16 는 Af 등방성 산란에 대 한 0-1 (병렬 중성자 산란 또는 자기장 방향 bicelles의 수직 정렬)에서 배열 한다.<a href="//ecsource.jove.com/files/ftp_upload/56812/56812fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
Bicelles의 구조는 다양 한 특성화 기법에 의해 광범위 하 게 연구 되었습니다. 13 자기 필드에 노출 하는 bicelles의 맞춤 NMR 분광학 또는 작은 각 중성자 산란 (SANS) 실험을 사용 하 여 측정할 되었습니다. 5 , 10 , 11 , 12 , 13 , 16 , 17 , 18 , 19 , 24 , 그러나 25 , 변화와 존재 Ln3 + 발생 NMR 봉우리의 확대는 메서드에 심각한 한계. 15 , 26 , 27 , 28 있지만 SAN 실험이이 제한, 대체에서 고통을 하지 않습니다 그리고 더 접근 기술 솔루션에서 어셈블리의 자석으로 유도 맞춤의 일상적인 정량화에 대 한 바람직한. 복굴절 측정 실용적이 고 비교적 간단한 대안입니다. 비슷하게 NMR 실험, 복굴절 측정 지질 재배열 및 지질 단계는 bilayer에 발생에 대 한 귀중 한 정보를 공개. 또한, 온도 같은 환경 조건 변경 polymolecular 어셈블리에서 발생 하는 기하학적 변환 모니터링 된다. 11 , 12 , 13 , 16 자기 유도 복굴절 Δn′ 인지질 시스템의 다양 한 유형을 공부에 사용 되었습니다. 13 , 29 , 30 는 자기장에서 위상 변조 기술에 따라 복굴절 측정 bicelles의 방향을 감지 하는 가능한 방법입니다. 12 , 16 , 18 , 29 , 31 , 32 35 T까지 높은 자기장에서 복굴절와 함께 bicelles를 조사 하는의 가능성 또한 M. Liebi 그 외 여러분 에 의해 입증 되었다 13
편광 이방성 물자 들어가면 일반 및 특별 한 파에서 굴절 됩니다. 11 두 파 다른 속도 있고 지체 δ에 의해 단계에서 이동 된다. 지체 δ의 정도 측정 하 고 복굴절 신호로 변환 <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> 는 소재를 사용 하 여 이방성의 정도 계량 하기
<img alt="Equation 6" src="/files/ftp_upload/56812/56812eq6.jpg">(2)
여기서 λ는 레이저와 d의 파장은 샘플의 두께입니다. 인지질은 광학 이방성 그리고 탄화수소 꼬리에 평행 하 게 그들의 긴 분자 축과 일치 하는 그들의 광 축. 11 , 12 는 인지질은 임의로 솔루션에서 지향 하는 경우 지체 없이 측정 됩니다. 지체는 인지질 서로 평행 정렬 됩니다 때 측정 된다. 자석으로 유도 복굴절 <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> ; 자기장에서 분자의 방향에 따라 양수 또는 음수 부호를 가질 수 있습니다 그림 2를 참조 하십시오. X 축에 평행 정렬 인지질 네거티브 귀 착될 것 이다 <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img>z 축을 따라 정렬 된 결과 긍정적인 그러나, <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img>. 아니 복굴절 때 광 축과 일치 빛 전파의 방향으로는 인지질 y 축에 평행 하 게 정렬 관찰 됩니다.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56812/56812fig2.jpg"> 
그림 2: 맞춤 인지질 및 자기 유도 복굴절의 해당 표시의 <img alt="Equation 12" src="/files/ftp_upload/56812/56812eq12.jpg"> </img> . 측정된의 <img alt="Equation 12" src="/files/ftp_upload/56812/56812eq12.jpg"> 는 자기장에서 인지질의 방향에 따라 달라 집니다. 파선 분자의 광 축을 나타냅니다. 빛은 45 °에서 편광 하 고 y 방향으로 전파. 자기장 B z 방향에서입니다. 이 그림은 M. Liebi에서 수정 되었습니다. 11 <a href="//ecsource.jove.com/files/ftp_upload/56812/56812fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
Bicelles의 등방성 콜 로이드 정지, 경우는 bilayer에 인지질의 배열에 의해 유도 된 방향 지체 δ 비우기 손실 될 것입니다. Bicelles는 또한 편광된 빛의 지체 δ를 일으키는 그들의 bilayers에 광학 활성 인지질을 찾으시는 순서 정렬 해야 합니다. 따라서, 복굴절 polymolecular 어셈블리의 자석 alignability 계량 하는 중요 한 도구입니다. Bicelles 정렬 자기장에 수직 긍정적인 보장할 <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img>병렬 정렬 그 부정적인 항복 것입니다 하는 동안, <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img>. 서명 설정의 정렬에 따라 달라 집니다 그리고 참조 샘플을 확인할 수 있습니다.

<table><tbody><tr><td>1,2-dimyristoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DMPC)</td><td>Avanti Polar Lipids</td><td>850345P</td><td>&gt;99%</td></tr><tr><td>1,2-dimyristoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate acid hexammonium salt (DMPE-DTPA)</td><td>Avanti Polar Lipids</td><td>790535P</td><td>&gt;99%</td></tr><tr><td>Thulium(III) chloride</td><td>Sigma-Aldrich</td><td>439649</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Dysprosium(III) chloride</td><td>Sigma-Aldrich</td><td>325546</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Ytterbium(III) chloride</td><td>Sigma-Aldrich</td><td>439614</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>319988</td><td>contains ethanol as stabilizer, ACS reagent, &amp;ge;99.8%</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>&amp;ge;99.9%</td></tr><tr><td>Cholesterol</td><td>Amresco</td><td>433</td><td>Ultra pure grade</td></tr><tr><td>D2O</td><td>ARMAR chemicals</td><td>1410</td><td>99.8 atom % D</td></tr><tr><td>Ultrapure water</td><td>Millipore</td><td></td><td>Synergy pak2 (SYPK0SIX2), Millipack GP (MPGP02001)</td></tr><tr><td>electronic pH meter</td><td>Metrohm</td><td>17440010</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 100nm membrane filter</td><td>VWR</td><td>515-2028</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 200nm membrane filter</td><td>VWR</td><td>515-2029</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 400nm membrane filter</td><td>VWR</td><td>515-2030</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 800nm membrane filter</td><td>VWR</td><td>515-2032</td><td></td></tr><tr><td>Whatmann Filter paper</td><td>VWR</td><td>230600</td><td></td></tr><tr><td>25 ml round bottom flask</td><td>VWR</td><td>201-1352</td><td>14/23 NS</td></tr><tr><td>3 ml glass snap-cup</td><td>VWR</td><td>548-0554</td><td>ND18, 18x30mm</td></tr><tr><td>2.5 ml glass syringe</td><td>Hamilton</td><td></td><td></td></tr><tr><td>Sodium dihydrogen phosphate dihydrate</td><td>Merk</td><td>1.06342</td><td>Salt used to make phosphate buffer</td></tr><tr><td>di-Sodium hydrogen phosphate</td><td>Merk</td><td>1.06586</td><td>Salt used to make phosphate buffer</td></tr><tr><td>Liquid Nitrogen</td><td>Carbagas</td><td>-</td><td></td></tr><tr><td>Pressurized Nitrogen gas</td><td>Carbagas</td><td>-</td><td>200 bar bottle</td></tr><tr><td>Lipid Extruder 10 ml</td><td>Lipex</td><td>-</td><td>Fully equipped with thermobarrel</td></tr><tr><td>High-pressure PVC tube</td><td>GR NETUM</td><td>-</td><td>must resist more than 4 MPa</td></tr><tr><td>Serto adaptors</td><td>Sertot</td><td>-</td><td></td></tr><tr><td>Nitrile gloves</td><td>VWR</td><td>-</td><td></td></tr><tr><td>2 ml glass pipettes</td><td>VWR</td><td>612-1702</td><td>230 mm long</td></tr><tr><td>Diode Laser</td><td>Newport</td><td>LPM635-25C</td><td></td></tr><tr><td>DSP Dual Phase Lock-in Amplifier</td><td>SRS</td><td>SR830</td><td></td></tr><tr><td>Photodiode Detector</td><td>Silonex Inc.</td><td>SLSD-71N5</td><td>5mm2, Silicon, photo-conductive</td></tr><tr><td>5.5 T Cryogenic Magnetic</td><td>Cryogenic/Oerlikon AG</td><td>-</td><td>12 bar He-cooled. RW4000/6000 compressor, RGD 5/100 TA cryo-head</td></tr><tr><td>Second order low pass filter</td><td>home-built</td><td>-</td><td>Linear power supply 24V DC, second order, Sallen Key, cut-off frequency 360 Hz, +/- 12V, max 10 mA</td></tr><tr><td>Photoelastic modulator</td><td>Hinds instruments</td><td>PEM-90</td><td></td></tr><tr><td>Glan-Thompson Calcite Polarizer</td><td>Newport</td><td>10GT04</td><td>25.4mm diameter</td></tr><tr><td>Quartz sample cuvette</td><td>Hellma</td><td>165-10-40</td><td>temperature controlled cell, 0.8 ml, 10mm path length</td></tr><tr><td>Temperature probe</td><td>Thermocontrol</td><td>-</td><td>Type K, 0.5mm diameter, Thermocoax</td></tr><tr><td>Non-polarizing mirrors</td><td>Newport</td><td>50326-1002</td><td>25.4mm</td></tr><tr><td>RS 232 cables</td><td>National Instruments</td><td>189284-02</td><td>For Connecting to the RS-232 Port on the front of Compact FieldPoint Controllers</td></tr><tr><td>BNC 50 &amp;Omega; cable and connectors</td><td>National Instruments</td><td>763389-01</td><td></td></tr><tr><td>cFP-AI-110</td><td>National Instruments</td><td>777318-110</td><td>8-Channel Analog Voltage and Current Input Module for Compact FieldPoint</td></tr><tr><td>cFP-CB-1</td><td>National Instruments</td><td>778618-01</td><td>Integrated Connector Block for Wiring to Compact FieldPoint I/O</td></tr><tr><td>cFP-CB-3</td><td>National Instruments</td><td>778618-03</td><td>Integrated Isothermal Connector Block for Wiring Thermocouples to the cFP-TC-120 Module</td></tr><tr><td>cFP-TC-120</td><td>National Instruments</td><td>777318-120</td><td>8-Channel Thermocouple Input Module for Compact FieldPoint</td></tr><tr><td>cFP-1804</td><td>National Instruments</td><td>779490-01</td><td>Ethernet/Serial Interface for NI Compact FieldPoint</td></tr><tr><td>LabView 2010</td><td>National Instruments</td><td>-</td><td></td></tr><tr><td>Industrial power supply</td><td>Traco Power</td><td>TCL 060-124</td><td>100-240V AC</td></tr><tr><td>Waterbath</td><td>Julabo</td><td>FP40-HE</td><td>refrigerated/Heating Circulator</td></tr></tbody></table>

fabrication procedures and birefringence measurements for designing magnetically responsive lanthanide ion chelating phospholipid assemblies

Bicelles는 조정 디스크 같은 polymolecular 어셈블리 지질 혼합물의 큰 다양성에서 형성. 응용 프로그램 범위에서 핵 자기 공명 (NMR)에 의해 막 단백질 구조 연구 nanotechnological 개발 광학 활성 및 자석으로 전환 젤의 형성을 포함 하 여. 이러한 기술 어셈블리 크기, 자석 응답 및 열저항의 높은 관리가 필요합니다. 1, 2의 혼합물-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)와 인지질 공액, 1, 2를 킬레이트 화는 란타넘족 이온 (Ln3 +)-dimyristoyl-sn-glycero-3-인-방법-신 triaminepentaacetate ( DMPE-DTPA), DMPC/DMPE-DTPA/Ln3 + 같은 높은 자석으로 응답 어셈블리에 조립 (어 금 니 비율 4:1:1) bicelles. 콜레스테롤 (철-오)과 독특한 physico-화학 특성을 제공 하는 어셈블리의 다른 세트에서 bilayer 결과에 스테로이드 파생 상품의 소개. 주어진된 지질 구성에 대 한 자석 alignability bicelle 크기에 비례 이다. Ln3 + 의 complexation 크기 및 정렬 방향 측면에서 전례 없는 자석 응답에 있는 결과. 난방 시 소포에 디스크와 같은 구조의 온도 가역 붕괴 정의 된 기 공 크기와 멤브레인 필터를 통해 압출 하 여 어셈블리의 치수의 조정 수 있습니다. 자석으로 alignable bicelles 기 선구자에 의해 정의 하는 어셈블리 차원에서 발생 하는 5 ° C에 냉각 하 여 다시 생성 됩니다. 여기, 그리고 어셈블리의 자석 alignability 5.5 T 자기장에서 복굴절 측정 하 여 정량이 제작 절차 설명 했다. 인지질 bilayer에서 발생 한 복굴절 신호는 더는 bilayer에서 발생 하는 polymolecular 변경 모니터링 수 있습니다. 이 간단한 기술은 이다 bicelles 하 고용 일반적으로 NMR 실험을 보완.

압출 비 DMPC/DMPE-DTPA/Tm3 + 의 복굴절 신호 (어 금 니 비율 4:1:1) 샘플 난방 및 냉각 주기 5에서 ~ 40 ° C 및 1 ° C/min (그림 6)의 속도로 다시 동안 5.5 T 자기장에서 모니터링 했습니다. 복굴절 결과 1.5 x 10-5, 배나 강한 보고 압출 시스템의 값이 있는 5 ° C에서 높은 자기 정렬 확인. 6 , 7 , <sup ...

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Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

자석으로 높은 반응 란타넘족 이온 킬레이트 화 polymolecular 어셈블리에 대 한 제작 절차 되 게 됩니다. 마그네틱 응답 nanopore 세포 막을 통해 밀어 남에 의해 맞게 어셈블리 크기에 의해 결정 됩니다. 어셈블리의 마그네틱 alignability 및 구조적인 변화 온도 유도 된 복굴절 측정, 핵 자기 공명 및 작은 각 중성자 뿌리기 무료 기법에 의해 감시 된다.

제작 절차 및 복굴절 측정 자석으로 응답 란타넘족 이온 킬레이트 화 인지질 어셈블리 설계에 대 한

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural ...

fabrication-procedures-birefringence-measurements-for-designing

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

7.1K 조회수.  ETH Zurich. 자석으로 높은 반응 란타넘족 이온 킬레이트 화 polymolecular 어셈블리에 대 한 제작 절차 되 게 됩니다. 마그네틱 응답 nanopore 세포 막을 통해 밀어 남에 의해 맞게 어셈블리 크기에 의해 결정 됩니다. 어셈블리의 마그네틱 alignability 및 구조적인 변화 온도 유도 된 복굴절 측정, 핵 자기 공명 및 작은 각 중성자 뿌리기 무료 기법에 의해 감시 된다.

비디오: Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

사이트 감독 스핀 라벨 및 펜타 머 리간드 - 게이 티드 이온 채널의 EPR 분광 연구

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

연구

화학

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

분리 및 통합 규조류 Cyclotella Meneghiniana Thylakoid 지질으로 리에서에서 광 수확 안테나

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

생화학

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

인지질 단백질 Annexin a 2 석 영 공명 기를 사용 하 여 지원 되는 고체 지질 Bilayers에 바인딩의 바인딩 역학 공부 되는 낭비 적인 Microgravimetry

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

스퀴지 기반의 조립 방법과 생체막 마이크로 어레이의 형성

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

생체공학

Measuring Magnetically-Tuned Ferroelectric Polarization in Liquid Crystals

Materials showing coupling phenomena between magnetism and (ferro)electricity, i.e., magnetoelectric effects, have attracted a great deal of attention due to their potential applications for future device technologies such as sensors and storage. However, conventional approaches, which usually utilize materials containing magnetic metal ions (or radicals), have a major problem: only a few materials have been found to show the coupling phenomena at room temperature. Recently, we proposed a new approach to achieve room-temperature magnetoelectrics. In contrast to conventional approaches, our alternative proposal focuses on a completely different material, a &#34;liquid crystal&#34;, free from magnetic metal ions. In such liquid crystals, a magnetic field can be utilized to control the orientational state of constituent molecules and the corresponding electric polarization through magnetic anisotropy of the molecules; it is an unprecedented mechanism of the magnetoelectric effect. In this context, this paper provides a protocol to measure ferroelectric properties induced by a magnetic field, that is, the direct magnetoelectric effect, in a liquid crystal. With the method detailed here, we successfully detected magnetically-tuned electric polarization in the chiral smectic C phase of a liquid crystal at room temperature. Taken together with the flexibility of constituent molecules, which directly affects the magnetoelectric responses, the introduced method will serve to allow liquid crystal cells to acquire more functions as room-temperature magnetoelectrics and associated optical materials.

In this report, we present a protocol to examine direct magnetoelectric effects, i.e., induction of ferroelectric polarization by applying magnetic fields, in liquid crystals. This protocol provides a unique approach, supported by the softness of liquid crystals, to achieve room-temperature magnetoelectrics.

액정에 자석으로 조정 Ferroelectric 편광 측정

Materials showing coupling phenomena between magnetism and (ferro)electricity, i.e., magnetoelectric effects, have attracted a great deal of attention due ...

공학

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

용매를 이용한 지질 이중층 (SALB) 방법에 의해 생체막 제작

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

물 샘플에서 추적 금속 분석을위한 쌍극자를 이용한 고체 단계 추출 마이크로 칩의 제작

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

형광 기반 측정 인스파디딜세린/포스파디딜리노시톨 4-인산염 교환 막 간

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

지원되는 지질 이중층에 리간드 나노 클러스터의 배열

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

단일 분자 확산 및 폴리머가 밀집된 지질 막에서의 조립

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...