この作品は、NIHの助成金GM073197とRR025336によって部品で賄われていた。

メソ結晶化実験での典型的な概要は図1,2に示されています。プレ結晶LCP - FRAPアッセイはオプションですが、彼 ​​らは大幅に特に困難な膜タンパク質3の場合には、初期結晶化条件の検索プロセスを加速することができます。 1。 LCPにおけるタンパク質の再構成<ol><li>洗剤溶液への関心の膜タンパク質を精製し、約10にタンパク質/界面活性剤複合体を集中- 20 mg / mLの、洗剤の1,4を介して、集中しないように注意しながら。 </li><li>転送〜LCPホスト脂質（通常はモノオレイン）または40で1.5 mLのプラスチックチューブとインキュベートに脂質混合物25mgの° Cで数分間脂質が溶けるまで。 </li><li> 100μLガスタイトシリンジに注射器のカプラーを取り付けます。 </li><li>調節可能なボリュームのピペットを用いて溶融脂質とシリンジをロードする。シリンジ内の脂質の量を記録。 </li><li> / Vタンパク質ソリューション対脂質比2 / 3のタンパク質溶液と、別の100μLシリンジをロードする対</li><li>注射器のカプラーを介して一緒に両方のシリンジを接続してください。 </li><li>脂質の中間相が均一になるまで注射器は、前後に、カプラーの内側の針を介して脂質とタンパク質を移動するために交互にプランジャ押し込みます。 LCPは、機械的混合によって自発的に形成し、そしてタンパク質は、LCPの脂質二重層に再構成になります。 LCPの形成は、小角X線回折1を使用して 、可能であれば、クロス偏光板とを備えた顕微鏡で見たときに、その透明性とゲル状の整合性がとbirefringencyの不在によって検証、またはすることができます。 </li></ol> 2。 LCP - FRAPプリ結晶化アッセイ LCP - FRAPアッセイは、スクリーニングの条件3の様々な時にLCPの再構成膜タンパク質の拡散特性を測定するように設計されています。 LCPの膜タンパク質の長距離拡散が成功した結晶化に不可欠ですが、LCPの微細構造は、大規模なタンパク質やオリゴマータンパク質の凝集体の拡散を抑制します。 メソ結晶化実験での失敗の一般的な理由は、拡散の損失につながる高速タンパク質凝集です。それは、タンパク質の凝集挙動は、特定のタンパク質の構造、ホストの脂質とスクリーニングのソリューション3の組成に依存することが示されている。 <ol><li> 〜100 / 1のタンパク質/色素比で蛍光色素（Cy3または類似の）とタンパク質にラベルを付ける、未反応色素を除去し、約1 mg / mLとするタンパク質を集中。遊離アミンまたは遊離チオールのいずれかにラベルを付けます。遊離アミンをラベリングする際、主に遊離のN -末端にラベルを付ける7と7.5の間のpHを使用してください。アミノ酸ラベリングも脂質蛋白質2,3と同じ場所に精製されたラベルを付けることができることに注意してください。 </li><li>再構成するセクション1で説明したようにLCPで標識したタンパク質を）。 </li><li>セクション3に記載されているようではなく、結晶化スクリーン2のLCP - FRAPスクリーニングのソリューションを使用して）アッセイプレートを設定します。 </li><li> 20℃でプレートをインキュベートします平衡状態を達成するには、少なくとも12時間、暗所でC。 </li><li>よく10倍対物レンズを用いて初のオンLCP - FRAPステーションと焦点上プレートのある場所。 </li><li>初期プリ漂白状態をキャプチャするために5蛍光画像を取得する。 </li><li>レーザーをトリガします。漂白スポットの真ん中に標識タンパク質の70％ - パルスのレーザパワーと番号は、漂白剤〜30に調整する必要があります。 </li><li>すぐにレーザーをトリガーした後、可能な限り高速なレートで約200画像の高速なポスト漂白シーケンスの記録を開始します。 </li><li>タンパク質の拡散速度に応じて、1〜20のように画像間の遅延を選択する、〜50枚の遅いポスト漂白シーケンスの録音で従ってください。 </li><li>すべてのフレームに漂白剤スポットの内部強度を統合し、レーザー漂白エリアの外の参照点の平均強度で漂白スポットの内部強度を割ることにより、集録中に漂白し、光強度の揺らぎのためにそれを修正する。 </li><li> 1に等しい事前に漂白強度は0に等しい初期漂白強度を確認するために修正された強度を正規化する。 </li><li>以下の式5を使用して 、正規化強度対時間、F（t）の曲線を合わせる： F（T）= M X EXP（- 2T / T）×（I 0（2T / T）+ I 1（2T / T））、（Eq.1） Mは拡散分子のモバイル割合である、Tは特徴的な拡散の時間、tはそれぞれの記録されたフレームのリアルタイムです、I 0とI 1 0 番と1 番目のオーダー変形ベッセル関数である。 </li><li>として、拡散係数、Dを計算します。 D = R 2 / 4T、（Eq.2） ここで、Rは、漂白スポットの半径である。 </li><li> tを移動する次もと繰り返します2.5）O - 2.13）。 </li><li>別のスクリーニングの条件で得携帯フラクションと拡散係数を比較する。デザインの新しい結晶化は、タンパク質の拡散とタンパク質の拡散が観察されなかったため除く条件を容易にするコンポーネントに基づいて選別します。タンパク質がスクリーニングの条件のいずれかに拡散しなかった場合、スクリーニングのスペースを広げたり、新しいタンパク質が構築しようと検討してください。 </li></ol> 3。 LCP結晶化トライアルのセットアップ<ol><li>再構成するセクション1で説明したようにLCPのタンパク質を）。 </li><li>タンパク質を含んだ反復シリンジディスペンサーに取り付けられた10μLガスタイトシリンジにLCPを転送します。 </li><li> 10μLシリンジに短いリムーバブルニードル（ゲージ26、10 mmの長さ）を取り付けます。 </li><li> 2x2の正方形を形成する4つの隣接する井戸の表面にLCPの200 NLのボーラス投与を分注する。 </li><li>対応する結晶の画面溶液1μLを持つLCPボーラス投与のそれぞれを重ねます。 </li><li>キャップの4つは18形のガラスのカバースリップをウェルの中にロードされます。井戸をシールするためにカバースリップ上に静かに押し込みます。 </li><li>プレート全体がいっぱいになるまで、4ウェルの次のセットで）手順3.4）-3.6を繰り返します。 </li><li>定期的に結晶の形成と成長のためのチェック、一定温度でインキュベートする。 </li></ol> 4。 LCPから収穫結晶<ol><li>直線、回転偏光子と検光子を装備した可変ズームとステレオ顕微鏡、下のタンパク質結晶プレートを置きます。 </li><li>全体がよくビューのフィールド内に配置されるように、低消費電力のズームを使って興味のあるもに焦点を当てる。 </li><li>セラミックキャピラリー切削石の鋭い角を使用しても境界線の内側の正方形を作成するための4ストロークでカバーガラスを得点する。 </li><li>カバーガラスの厚さによって傷を伝播する強力な鋭い点ピンセットで獲得した周囲に押してください。 </li><li>得点の正方形の対角線上にあるパンチ二つの小さな穴。 </li><li>後続の手順中に脱水症状を減らすために穴のいずれかを使用して沈殿剤溶液を数μLを注入する。 </li><li>角度の鋭い針のプローブを使用すると、カットアウトの正方形を解放する1つまたは2つの側面に沿ってガラスを破る。 </li><li>慎重にキュービック相のボーラスを監視ガラスの正方形を持ち上げます。ボーラスがカバースリップに付着されている場合は、井戸の底にガラスの上の正方形と場所を反転させる。 </li><li>必要に応じてウェル内の露出キュービック相のボーラスの上に、、凍結保護剤を添加した沈殿剤溶液中の余分な数μLを、追加。 </li><li>顕微鏡の倍率を増加させると、結晶に焦点を当てる。 </li><li>収穫のループを参照するのに十分な光を保ちながら、偏光板と複屈折性結晶と背景のコントラストを高めるために、アナライザの間の角度を調整します。 </li><li>結晶サイズに一致する直径MiTeGen顕微標本を選択し、顕微標本にそれをすくうことによりLCPから直接結晶を収穫。 </li><li> Flashは、液体窒素で収穫した結晶と顕微標本を凍結し、X線データ収集6のシンクロトロン光源のビームラインに発送して下さい。 </li></ol> 5。代表的な結果： 人工ヒトβ2アドレナリンGタンパク質共役型受容体（β2 AR - T4Lは）バキュロウイルスSf9昆虫細胞に感染し、部分的インバースアゴニストカラゾロール7に束縛dodecylmaltoside（DDM）/コレステヘミサ（CHS）洗剤溶液で精製で発現させた。タンパク質をCy3 NHSエステルで標識し、LCP - FRAPプリ結晶アッセイ（図2）で使用されていました。 LCP - FRAPアッセイの結果から選ばれたいくつかの条件に基づいて、粗いグリッドの画面は、初期の結晶のようなヒット（図3）を製作。沈殿剤条件のさらなる最適化は、回折品質の結晶（図4）を得た。 <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="図1" /> 図1典型的なLCPの結晶化実験のフローチャート。グレーのボックス内のステップは、現在のプロトコールに記載されていません。 <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="図2" /> モノオレイン基づくLCPのβ2 AR-T4L/carazololと図2。LCP - FRAPアッセイ。 A）LCP - FRAPアッセイの結果は、96ウェルプレートの各サンプルを順番に漂白され、蛍光の回復が30分間のインキュベーション後に測定される、自動ハイスループットモードで実行。各試料におけるモバイル分数を表す得られる蛍光の回復は、すべて96サンプルのためにプロットされます。スクリーニングのソリューションは、2つの異なる濃度で48の異なる塩と組み合わせて、0.1 MトリスpHを8、30％（v / v）のPEG 400が含まれています。 B）複数の担当者のための蛍光回復プロファイルresentative条件。実線の曲線は、式による近似を表す。 1。モバイルフラクションと拡散係数は、式を用いて決定されます。 1と2。ナトリウム塩化物を含む試料中の10％未満の高速リカバリは、蛍光標識脂質タンパク質と共精製さによるものです。 <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="図3" /> 図3β2 AR-T4L/carazololの初期結晶のヒットは、硫酸Naは含まれている、LCP - FRAPによって識別された最も有望な条件の周りに粗いグリッドのスクリーニングによって得られた（パネル）およびNaギ（パネルB）。タンパク質をCy3 NHSエステルで標識し、蛍光画像は、605 nmで543 nmで励起し、発光を用いて撮影されています。 <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="図4" /> 図4β2 AR-T4L/carazololの最適化された結晶。ナトリウム硫酸塩の存在（パネルAおよびB）で成長した結晶とKギ（パネルCおよびD）の画像は、明視野モード（パネルAおよびC）で撮影し、クロス偏光子を（パネルBおよびD）を使用している。

<ol>
	<li>Caffrey, M., 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=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat. Protoc. 4, 706-731 (2009).</li><li>Cherezov, V., Abola, E., 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=Recent+progress+in+the+structure+determination+of+GPCRs,+a+membrane+protein+family+with+high+potential+as+pharmaceutical+targets.">Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets.</a> Methods Mol. Biol. 654, 141-170 (2010).</li><li>Cherezov, V., Liu, J., Hanson, M. A., Griffith, M. T., 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=LCP-FRAP+assay+for+pre-screening+membrane+proteins+for+in+meso+crystallization.">LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization.</a> J. Cryst. Growth Design. 8, 4307-4315 (2008).</li><li>Misquitta, Y., 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=Detergents+destabilize+the+cubic+phase+of+monoolein:+implications+for+membrane+protein+crystallization.">Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization.</a> Biophys. J. 79, 394-405 (2003).</li><li>Soumpasis, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+fluorescence+photobleaching+recovery+experiments.">Theoretical analysis of fluorescence photobleaching recovery experiments.</a> Biophys. J. 41, 95-97 (1983).</li><li>Cherezov, V., Hanson, M. A., Griffith, M. T., Hilgart, M. C., Sanishvili, R., Nagarajan, V., Stepanov, S., Fischetti, R. F., Kuhn, P., 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=Rastering+strategy+for+screening+and+centering+of+microcrystal+samples+of+human+membrane+proteins+with+a+sub-10+micrometer+size+X-ray+synchrotron+beam.">Rastering strategy for screening and centering of microcrystal samples of human membrane proteins with a sub-10 micrometer size X-ray synchrotron beam.</a> J. R. Soc. 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>

利害の衝突は宣言されません。

プロトコルは、 メソ結晶化実験での実行に関連する主要な手順のための基本的な視覚的なガイダンスを提供します。深いこれらのプロトコルに関連する詳細、可能な落とし穴、欠点または代替ルートを強調するよりは、他の場所で1,2用意されています。オプションのLCP - FRAPアッセイは、最も有望なタンパク質の構造を選択するLCPのホストの脂質および脂質添加物だけでなく、可能な沈殿剤と緩衝液の条件3の範囲を制限するために以前の段階で役立ちます。初期結晶化ヒットが検出されると、それはより品質の高い結晶を得るために最適化する必要があります。 メソ結晶化条件での最適化は、LCP 1の組成物に関連付けられている余分なパラメータを追加することで可溶性タンパク質のための条件を最適化すると本質的に似ています。脂質中間相で成長した膜タンパク質の結晶は、通常、界面活性剤溶液中で得られた結晶よりも小型ですが、より現代的な放射光源6でマイクロフォーカスを使用してから強く恩恵を受け、そのため、利用できるビームライン命じた。 、結晶化またはアッセイプレートを設定するLCP - FRAPアッセイを行うと、結晶の検出を含むメソ結晶化、、 に関連する手順の多くは、できるように、1,2,8,9半または完全に自動化されている条件の広い範囲のスクリーニングタンパク質と脂質の消費する少量ながら。一方、LCPと結晶の収穫におけるタンパク質reconstitutionsは、マニュアルや面倒な操作が残ると、このように、改善の必要性を持っている。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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

膜タンパク質は、形質導入、輸送、エネルギー変換を信号に関係する生体細胞内の重要​​な機能を実行し、そして、そのように、故障や病気の多くに関与している。しかし、膜タンパク質の構造と機能の理解が強く、主に、それらの水溶性パートナーのその背後にそれらの可溶化と回折品質結晶の生成に関連する問題のために遅れている。脂質中間相の結晶化は、（また、 メソ or LCP結晶でも呼ばれる）が正常に微生物ロドプシン、光合成タンパク質、外膜のβバレルとGタンパク質共役受容体の高分解能構造を得るために適用was有望な技術です。メソで結晶化は、ネイティブのような膜環境を活用し、一般的に低い溶媒含量と洗剤のソリューションから、従来の結晶に比べて、よりよい順序で結晶を生成します。方法は難しくありませんが、粘性の中間相の材料を処理する際の脂質の相挙動と実践を理解する必要があります。ここでは、事前に結晶化アッセイとから収穫結晶を行い、LCPを作成し、アッセイまたは結晶化プレートにLCPのナノリットルの一部を分配することによって続いて注射器のミキサーを使用してLCPの脂質二重層の膜タンパク質を、再構成のシンプルかつ効率的な方法を示していますLCPの行列。これらのプロトコルは、 メソ結晶化試験で近づくための基本的なガイドを提供しますが、任意の結晶化実験と同様に、広範なスクリーニングと最適化が必要であり、成功した結果は必ずしも保証されません。

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

Crystallization of Membrane Proteins in Lipidic Mesophases

プロトコルは、LCP結晶化試験と収穫結晶の設定、LCP - FRAPプリ結晶アッセイで初期条件を見つけることが、脂質キュービック相（LCP）におけるタンパク質の再構成から開始して膜タンパク質の回折品質結晶を得るために不可欠な手順を説明します。 。

脂質中間相における膜タンパク質の結晶化

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 ビュー.  The Scripps Research Institute. プロトコルは、LCP結晶化試験と収穫結晶の設定、LCP - FRAPプリ結晶アッセイで初期条件を見つけることが、脂質キュービック相（LCP）におけるタンパク質の再構成から開始して膜タンパク質の回折品質結晶を得るために不可欠な手順を説明します。 。

ビデオ: 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.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

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

生物学

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.

電子線結晶学による構造生物学研究用小型膜タンパク質の二次元結晶化トライアルの評価

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.

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

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.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

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.

脂質中間相における膜タンパク質のハイスループット結晶化のためのロボットの使用

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.

高分子結晶学による構造決定のための脂質メソフェーズで栽培膜タンパク質の収穫とクライオ冷却結晶

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.

蛍光タンパク質を発現する生きたマウスでのイメージング細胞内の構造物の生体顕微鏡

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.

単離されたミトコンドリアのフローサイトメトリーによる外膜タンパク質の免疫検出

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.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</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.

ラット腸間膜に微小血管透過性の調節を調査するために、微小灌流法

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