이 작품은 NIH 보조금 GM073197 및 RR025336의 부품에 투자했다.

meso의 결정화 실험에의 일반적인 개요는 Fig.1 1,2로 표시됩니다. 사전 결정 LCP - FRAP의 assays은 선택 사항입니다 있지만, 그들은 크게 특히 어려운 막 단백질 3의 경우, 초기 결정화 조건 검색하는 과정을 가속하실 수 있습니다. 1. LCP의 단백질 Reconstitution <ol><li> 세제 솔루션에 대한 관심의 막 단백질을 정화하고 ~ 10 단백질 / 세제 단지 집중 - 20 MG / ML을 통해 - 집중 세제 1,4에 관심을 찍어. </li><li> 전송 ~ LCP 호스트 지질 (일반적으로 monoolein) 또는 40 1.5 ML 플라스틱 관과 부화에 지질 혼합물의 25 MG ° C 몇 분 동안 지방질이 녹으까지. </li><li> 100 μL 가스 기밀 주사기에 주사기의 커플러를 연결합니다. </li><li> 조정 볼륨 피펫을 사용하여 용융 지질과 주사기를로드합니다. 주사기에있는 지방질의 볼륨을 기록합니다. </li><li> 단백질 솔루션 - 투 - 지질 비율 3분의 2 V /에서 단백질 솔루션으로 앞으로 100 μL 주사기를 로드할 대 </li><li> 주사기의 커플러를 통해 함께 두 주사기를 연결합니다. </li><li> 지질 mesophase가 균질 될 때까지 주사기가 앞뒤로, 커플러의 내부 주사 바늘을 통해 지방질과 단백질을 이동 번갈아 plungers 누르십시오. LCP는 기계적 혼합시 자발적으로 형성하고, 단백질은 LCP의 지질 이중층에 재구성된다. LCP의 형성은 작은 각도 X - 선 회절 1을 사용하여 가능하면 교차 polarizers 장착된 현미경으로 볼 때 그 투명성과 겔형 일관성에 의해 및 birefringency의 부재에 의해 검증, 또는 수 있습니다. </li></ol> 2. LCP - FRAP 사전 결정화 Assays LCP - FRAP의 assays는 심사 조건 3 다양한에서 LCP의 재구성 막 단백질의 확산 특성을 측정하기 위해 고안되었습니다. LCP의 막 단백질의 장거리 확산은 성공적인 결정을 위해 필수적입니다 있으나, LCP의 미세 구조는 큰 단백질이나 oligomeric 단백질 집계의 확산을 constrains. meso의 결정화 실험에서 실패를위한 일반적인 이유는 확산의 손실로 이어지는 빠른 단백질 집합입니다. 이것은 단백질의 집계 문제는 특정 단백질 구조, 호스트 지질 및 심사 솔루션 3 구성에 따라 달라집니다 것으로 나타났습니다. <ol><li> ~ 1분의 100의 단백질 / 염료 비율에서 형광 염료 (Cy3 또는 이와 유사한 것)와 단백질을 분류, unreacted 염료를 제거하고 ~ 1 MG / ML에 단백질을 집중. 레이블 중 무료 아민 또는 무료 thiols. 무료 아민을 라벨 때, predominately 자유 N - 말단 레이블을 7과 7.5 사이의 산도를 사용합니다. 아미노산 라벨은 또한 lipids 단백질 2,3 공동 정화 레이블을 수있다는 점에 유의하십시오. </li><li> Reconstitute로 제 1에서 설명한 LCP의 표시 단백질). </li><li> 대신 결정화 화면 2 LCP - FRAP 검사 솔루션을 사용) 제 3에서 설명한대로 분석 번호판을 설정합니다. </li><li> 20 접시를 품어 ° 평형 상태를 달성하기 위해 최소한 12 시간 동안 어둠 속에서 C. </li><li> 10X 목표를 사용하여 잘 첫 LCP - FRAP 기차역과 집중 플레이스 접시 중 하나를. </li><li> 처음 이전 표백 상태를 캡처 5 형광 이미지를 취득. </li><li> 레이저를 트리거. 표백 지점의 중앙에 표시된 단백질의 70 % - 펄스의 레이저 전원과 전화 번호는 표백제 ~ 30으로 조정해야합니다. </li><li> 바로 레이저를 실행 후, 가장 빠른 속도로 ~ 200 이미지의 빠른 사후 표백 시퀀스를 녹음 시작합니다. </li><li> 단백질의 확산 속도에 따라 10-20의 이미지로 사이의 지연 시간을 선택 ~ 50 이미지의 느린 후 표백제 순서의 기록과 함께하십시오. </li><li> 모든 프레임에있는 표백제 지점 내부의 강도를 통합하고 레이저 표백 영역 밖의 참조 지점의 평균 농도로 표백 현장 내부의 강도를 나누어 수집하는 동안 강도 변동을 표백과 빛을 위해 수정. </li><li> 1 같은 사전 표백 강도와 0으로 동등한 초기 표백 강도를 확인하기 위해 수정된 강도를 정상화. </li><li> 다음 방정식 5를 사용하고, 표준 강도 대 시간, F (t)의 곡선에 맞게 : F (t) = M X EXP (- 2T / T) × (I 0 (2T / T) + I 1 (2T / T)), (Eq.1) M은 diffusing 분자의 모바일 분수이고, T는 특성 확산 시간, T는 전 0 각 기록된 프레임의 실시간이며 1 0 회와 베셀 함수를 수정 제 1 순서입니다. </li><li> 확산 계수, D를 같이 계산 : D = R 2 / 4T (Eq.2) 어디 R은 표백 지점의 반경이다. </li><li> t으로 이동2.13) - 다음 잘하고 단계를 반복 2.5) O. </li><li> 다른 심사 조건 얻은 모바일 분수와 확산 계수를 비교합니다. 디자인 새로운 결정은 단백질 보급과 단백질 확산이 관찰되지 않은에 대한 제외 조건을 촉진하는 구성 요소에 따라 화면. 단백질은 검사 조건에도 확산하지 않은 경우, 심사 공간을 확대하거나 새로운 단백질 구조하려고 생각합니다. </li></ol> 3. LCP 결정화 평가판 설정 <ol><li> LCP의 Reconstitute 단백질 등) 제 1에서 설명한. </li><li> 반복 주사기 자동 지급기에 부착된 10 μL 가스 기밀 주사기로 단백질 라덴 LCP을 전송합니다. </li><li> 10 μL 주사기로 짧은 이동식 바늘 (게이지 26, 10mm 길이)를 연결합니다. </li><li> 2x2 사각형을 형성 네 인접한 웰스의 표면에 LCP 200 NL의 boluses 하는걸. </li><li> 해당 결정화 화면 솔루션 1 μL와 LCP의 boluses 각 오버레이. </li><li> 18mm 정사각형 유리 coverslip와 모자 네로드 웰스. 우물을 봉인하기 위해 coverslip에 부드러운 압력을 적용합니다. </li><li> 전체 접시가 가득 때까지 네 우물의 다음 세트) 단계 3.4) -3.6를 반복합니다. </li><li> 주기적으로 크리스탈 형성과 성장에 대한 확인, 일정한 온도에서 접시를 품어. </li></ol> 4. LCP에서 결정 수확 <ol><li> 선형 편광 회전 및 분석기가 장착된 가변 줌과 스테레오 현미경으로 단백질 결정과 접시를 놓습니다. </li><li> 평생 잘가 볼 분야 내에서 배치되도록 낮은 전력 축소를 사용하여 관심있는 잘 중점을 둡니다. </li><li> 세라믹 모세관 절삭 석의 날카로운 모서리를 사용하여 잘 경계 안에 사각형을 만드는 네 뇌졸중의 coverslip 유리를 점수. </li><li> coverslip 유리의 두께를 통해 흠집을 전파하기 위해 강한 날카로운 점 핀셋으로 득점 주위에 누르십시오. </li><li> 점수 광장의 반대편 구석에 펀치 구멍이 작은. </li><li> 이후 단계 동안 탈수를 줄이기 위해 구멍 중 하나를 통해 침전제 솔루션의 몇 가지 μL를 주입. </li><li> 직각 날카로운 바늘 프로브를 사용하면 컷 아웃 광장을 무료로 하나 또는 두 개의 면을 따라 유리를하다. </li><li> 조심스럽게 입방 위상 볼러스에 대한 유리 스퀘어 시청을 올려요. 볼러스이 coverslip에 붙어있다면, 우물의 바닥에 유리를 통해 광장과 장소를 플립. </li><li> 와 보충 침전제 솔루션의 추가 몇 μL를 추가 cryo - protectant, 필요한 경우, 우물에 노출 입방 위상 볼러스 위에. </li><li> 현미경의 배율을 증가하고 수정하는 데 중점을 둡니다. </li><li> 수확 루프를 볼 수있을만큼 가벼운 유지하면서, 편광과 복굴절 결정과 배경 사이의 대비를 증가 분석기 사이의 각도를 조정합니다. </li><li> 크리스탈 크기를 일치 직경 MiTeGen MicroMount를 선택하고 다음 MicroMount에 그것을 떠서하여 LCP에서 직접 크리스탈을 수확. </li><li> 플래시 액체 질소의 수확 크리스탈 MicroMount을 동결하고, X - 레이 데이터 수집 6 싱크로 트론 소스 beamline로 보내주십시오. </li></ol> 5. 대표 결과 : 설계 인간 베타 2 아드레날린 G 단백질 결합 수용체 (β 2 AR - T4L)은 baculovirus sf9 곤충 세포를 감염 dodecylmaltoside (DDM) / 부분 역 작용제 carazolol 7 바운드 cholesteryl hemisuccinate (CHS) 세제 용액에 정화로 표현되었다. 단백질은 Cy3 NHS 에스테르로 표시하고 LCP - FRAP 미리 결정화 assays (그림 2)에 사용되었다. LCP - FRAP assays의 결과에서 선택한 여러 조건에 따라 거친 그리드 화면 초기 크리스탈처럼 조회 (그림 3)을 생산. 침전제 조건 추가 최적화 회절 품질 결정 (그림 4)를 굴복. <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="그림 1" /> 그림 1. 전형적인 LCP의 결정화 실험의 흐름 차트. 회색 상자에서 단계를 현재 프로토콜에서 설명하지 않습니다. <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="그림 2" /> monoolein 기반 LCP의 β 2 AR-T4L/carazolol과 그림 2. LCP - FRAP 분석. A) LCP - FRAP 분석 결과는 96 - 웰 플레이트의 각 샘플이 순차적으로 표백하고 형광 복구는 30 분 보육 후에 측정되는, 자동으로 높은 처리량 모드에서 수행했습니다. 각 예제에서 모바일 분수를 나타내는 얻은 형광 recoveries은 모든 96 샘플 꾸몄다 있습니다. 심사 솔루션은 두 가지 다른 농도 48 다른 소금과 함께 0.1 M 트리스 산도 8, 30 % V / V PEG 400이 포함되어 있습니다. B) 여러 세일즈맨을위한 형광 복구 프로필resentative 조건. 실선 곡선은 EQ로 맞는를 나타냅니다. 1.The 모바일 분수와 확산 계수는 Eqs를 사용하여 결정됩니다. 1 2. 나 염화가 들어있는 예제에서 10 % 미만의 빠른 회복은 단백질과 함께 공동 정화 찬란 표시 lipids 때문입니다. <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="그림 3" /> 그림 3. β 2 AR-T4L/carazolol의 초기 결정 조회수가 황산 나 포함, LCP - FRAP 식별 가장 유망 조건 주위에 굵은 격자 심사에 의해 얻은 (패널 A)과 나 편대 (패널 B). 단백질은 Cy3 NHS 에스테르로 표시되고 형광 이미지는 605 nm의에서 543 nm의 여기에서와 방사를 사용하여 촬영하고 있습니다. <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="그림 4" /> 그림 4. β 2 AR-T4L/carazolol의 최적화 결정. (패널 A와 B) 나 황산의 존재에서 재배 결정의 이미지와 K 편대는 (패널 C와 D) 브라이트 모드 (패널 A 및 C)에서 찍은 상호 polarizers을 (패널 B와 D)를 사용하고 있습니다.

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Interface. 6, S587-S597 (2009).</li><li>Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318, 1258-1265 (2007).</li><li>Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robotic+system+for+crystallizing+membrane+and+soluble+proteins+in+lipidic+mesophases.">A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.</a> Acta Crystallogr. D. 60, 1795-1807 (2004).</li><li>Kissick, D. J., Gualtieri, E. J., Simpson, G. J., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+imaging+of+integral+membrane+protein+crystals+in+lipidic+mesophases.">Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.</a> Anal. Chem. 82, 491-497 (2010).</li><li>Cheng, A., Hummel, B., Qiu, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+mechanical+mixer+for+small+viscous+lipid-containing+samples.">A simple mechanical mixer for small viscous lipid-containing samples.</a> Chem. Phys. Lipids. 95, 11-21 (1998).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+inexpensive+nanoliter-volume+dispenser+for+highly+viscous+materials+used+in+membrane+protein+crystallization.">A simple and inexpensive nanoliter-volume dispenser for highly viscous materials used in membrane protein crystallization.</a> J. Appl. Cryst. 38, 398-400 (2005).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-volume+plates+with+excellent+optical+properties+for+fast,+inexpensive+crystallization+screening+of+membrane+proteins.">Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins.</a> J. Appl. Cryst. 36, 1372-1377 (2003).</li><li>Cherezov, V., Fersi, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+screens:+compatibility+with+the+lipidic+cubic+phase+for+in+meso+crystallization+of+membrane+proteins.">Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins.</a> Biophys J. 81, 225-242 (2001).</li></ol>

관심 없음 충돌 선언하지 않습니다.

프로토콜은 meso의 결정화 실험의 수행에 관련된 기본 단계에 대한 기본적인 시각 지침을 제공합니다. 심층의 가능한 함정을 강조 이러한 프로토콜에 관한 세부 사항 더, 결점이나 대안 노선은 다른 1,2 사용할 수 있습니다. 옵션 LCP - FRAP의 assays은 가장 유망한 단백질 구조, LCP 호스트 지질 및 지질 첨가제뿐만 아니라, 가능한 precipitants 및 버퍼 조건 3 범위를 제한을 선택합니다 이전 단계에서 도움이 될 수 있습니다. 초기 결정화 히트가 발견되면, 그것은 더 나은 품질의 결정을 얻기 위해 최적화해야합니다. meso의 결정화 조건의 최적화는 LCP 1의 구성과 관련된 추가적인 매개 변수의 추가와 함께 가용성 단백질에 대한 조건을 최적화하기 위해 본질적으로 비슷합니다. lipidic mesophase 재배 막 단백질 결정은 일반적으로 세제 용액에서 얻어진 결정보다 크기가 작은,하지만, 더 많은 현대적인 싱크로 트론 소스 6 microfocus를 사용 강하게 benefitting, 따라서 가능한 beamlines 명령했다. , 결정 또는 분석 번호판을 설정 LCP - FRAP의 assays를 실시하고 결정을 감지 포함한 meso의 결정,에 관련된 절차의 대부분은 허용, 1,2,8,9 반 또는 완전히 자동되었습니다 조건의 큰 범위의 심사 단백질과 지방질의 소비 소량 동안. 한편, LCP 및 수정 수확의 단백질 reconstitutions 수동 더 지루한 작업을 유지하고, 따라서, 개선을위한 필요가있다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>100 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7656-01</td>
<td>2 syringes are required</td>
</tr>
<tr>
<td>10 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7653-01</td>
<td>&nbsp;</td>
<tr>
<td>Syringe coupler</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7770-02 30902</td>
<td>The coupler can be made using available parts as described in refs. 10</td>
</tr>
<tr>
<td>Repetitive syringe dispenser</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>83700</td>
<td>The repetitive syringe dispenser can be modified to reduce dispensing volume by ~3 times11</td>
</tr>
<tr>
<td>Short (0.375&#x201D;) flat-tipped removable needle (point style 3, gauge 26)</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7804-03</td>
<td>&nbsp;</td>
<tr>
<td>96-well glass sandwich plate</td>
<td>&nbsp;</td>
<td>Marienfeld</td>
<td>08 900 03</td>
<td>For manual operations it is more convenient to assemble the glass sandwich plate using a standard microscope glass slides and a double sticky tape with punched holes1,2,12.</td>
</tr>
<tr>
<td>Glass cover slip</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>63787-01</td>
<td>&nbsp;</td>
<tr>
<td>monoolein</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7765</td>
<td>&nbsp;</td>
<tr>
<td>Crystallization screens</td>
<td>&nbsp;</td>
<td>Hampton Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience</td>
<td>&nbsp;</td>
<td>Most of available commercial screens can be used for initial screening. Conditions that consistently disrupt LCP can be diluted 2x for better compatibility13.</td>
</tr>
<tr>
<td>Capillary cutting stone</td>
<td>&nbsp;</td>
<td>Hampton Research</td>
<td>HR4-334</td>
<td>&nbsp;</td>
<tr>
<td>Fine point tweezers</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>510</td>
<td>&nbsp;</td>
<tr>
<td>Angled sharp probe</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13650</td>
<td>&nbsp;</td>
<tr>
<td>MicroMounts</td>
<td>&nbsp;</td>
<td>MiTeGen</td>
<td>M1-Lxx-xx</td>
<td>Select MicroMount diameter to match the crystal size</td>
</tr>		</tbody>
		</table>
		</div>

crystallization of membrane proteins in lipidic mesophases

막 단백질은 전달, 운송 및 에너지 변환, 그리고 같은 고장과 질병의 다수 연루된다 신호에 관한 살아있는 세포에서 중요한 기능을 수행합니다. 그러나, 멤브레인 단백질의 구조와 기능 이해 강하게 인해 회절 품질 결정 자신의 solubilization 및 생성과 관련된 문제로, 주로 그들의 가용성 파트너의 뒤쳐지고있다. lipidic mesophases의 결정체 (또한 meso 또는 LCP 결정화에서라고도 함) 성공적으로 미생물 rhodopsins, 광합성 단백질, 바깥쪽 막 베타 두통 및 G 단백질 결합 수용체의 고해상도 구조를 얻기 위해 적용했던 ​​유망 기술입니다. meso에 결정은 네이티브처럼 막 환경을 활용하고 일반적으로 낮은 용매 컨텐츠 및 세제 솔루션에서 전통적인 결정에 비해 더 나은 주문과 수정을 생산하고 있습니다. 방법은 어려운 것은 아니지만 점성 mesophase 자료를 취급하는 지질 위상 행동과 실천의 이해를 필요로합니다. 여기 LCP을하고, 분석 또는 결정화 판에 LCP의 nanoliter의 일부를 분배 미리 결정화 assays를 실시하고으로부터 결정을 수확하여 다음 주사기 믹서를 사용하여 LCP의 지질 이중층의 막 단백질을 reconstituting의 간단하고 효율적인 방법을 입증 LCP 매트릭스. 단, 결정화 실험, 광범위한 검사 및 최적화와 마찬가지로 필요하며, 성공적인 결과는 반드시 보증하지 않습니다,이 프로토콜은 meso의 결정화 실험의 접근에 대한 기본적인 가이드를 제공합니다.

Watch this Scientific Journal Video about Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

Crystallization of Membrane Proteins in Lipidic Mesophases

프로토콜은 LCP 결정화 시련과 수확 결정을 설정, LCP - FRAP 미리 결정화 assays과 초기 조건을 찾는 lipidic 입방 단계 (LCP)에서 단백질의 reconstitution부터 막 단백질의 회절 품질 결정을 얻기 위해 필수적인 단계를 설명 .

Lipidic Mesophases의 막 단백질의 결정화

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

crystallization-of-membrane-proteins-in-lipidic-mesophases

Research

JoVE Journal

Biology

31.2K 조회수.  The Scripps Research Institute. 프로토콜은 LCP 결정화 시련과 수확 결정을 설정, LCP - FRAP 미리 결정화 assays과 초기 조건을 찾는 lipidic 입방 단계 (LCP)에서 단백질의 reconstitution부터 막 단백질의 회절 품질 결정을 얻기 위해 필수적인 단계를 설명 .

비디오: Crystallization of Membrane Proteins in Lipidic Mesophases

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidic Mesophases를 사용하여 구조 결정을위한 막 단백질 Crystallizing

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

연구

생물학

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

전자 Crystallography에 의해 구조 생물학 연구에 대한 작은 막 단백질의 2 차원 결정화 평가판을 평가

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

막 단백질의 Conformational 역학 검사 현장에서 사이트 이동 형광 라벨링과

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidic Mesophases의 막 단백질의 높은 처리량을 결정하기위한 로봇의 사용

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Macromolecular의 결정학에 의해 구조 결정에 대한 Lipidic Mesophases에서 재배 막 단백질의 수확 및 Cryo 냉각 크리스털

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

형광 단백질을 표현 라이브 마우스의 이미징 세포 이하의 구조물의 intravital 현미경

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

고립 된 미토콘드리아의 유동 세포 계측법에 의해 외부 막 단백질의 면역

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusion 기술은 쥐 장간막에서 미세 혈관 투과성의 규정을 조사하기 위해

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...