この作品は、ドイツ研究振興協会 (INST 39/969-1) し ERC 助成契約 n. 681891 を通じて欧州研究評議会によって資金を供給します。

<ol>
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複雑なタンパク質系のこれらの測定値の分析の手順を追って説明 3色 smFRET データを取得する実験を紹介します。このアプローチでは、直接複数の相互作用部位や構造変化との相関を評価するユニークな可能性を提供しています。
タンパク質の適切なマルチカラー分子データを得るために低ノイズ レベルで再現可能な測定を行うことが重要です。これは、フロー商工会議...

多くの蛋白質は、動的錯体の構造変化と幅広い時間スケール1,2,3の一時的な連合を介した他の分子とその機能を発揮します。外部のエネルギー源 (例えばATP) これらの動的相互作用することができます機能サイクルの方向性につながる、最終的に生活のための前提条件のセルに非平衡定常状態を維持する結合。
これらの分子機械を完全に理解するためには、静的な記述の構造研究に導かれては十分ではありません。さらに、基になる運動モデルの知識を持って運動速度定数を決定するために不可欠です。いくつかの既存の方法許可バイナリ相互関心、表面プラズモン共鳴など、(例えば、ジャンプまたはストップト フロー分光読み出しと緩和法の 2 つの分子のダイナミクスを研究する研究者テクニック) と核磁気共鳴。ただし、適用性は、(例えば、 1 つのバインドとバインドされていない状態) の単純な 2 状態系平均一括実験に固有のために制限されてほとんどの場合です。以上の国または中間体が関与している場合、彼らは唯一速度定数の複雑な混合物をもたらします。単一分子光ピンセットや磁気ピンセットや 2 色 smFRET、つまり1 ドナー表面固定化したサンプルを 1 つの受容体 fluorophore など方法は、立体配座の変化すべての速度定数を回復できます。しかしときに、1 つ以上の結合部位に影響を与える相互作用は、これらの方法が限られている残る、2 つ (またはそれ以上) の可能な相関相互作用についてのみ、一連の実験から結論は間接を通じてアクセスできます。
マルチカラー smFRET4,5,6,7,8,9は、リアルタイムで直接、下のこれらのコンポーネント間の相互作用を研究する機会を提供しています近く生理的条件10。これにより、たとえば、リガンドまたは別タンパク質8,9,11のコンホメーション依存的結合を調査する 1 つ。ここで示した全体的なアプローチは、測定チャンバーの表面にタンパク質を付けると時間をかけて (の詳細についてを参照してください9プリズム型観察上の蛍光強度を追跡するための特定の位置で興味のタンパク質のラベルには,12) します。 異なる染料の空間的近接性することができますそれらの間のエネルギー移動から決定されます。(文献13) 蛋白質蛋白質から異なる場合があります戦略をラベリングと smFRET 測定におけるアーティファクトを避けるためのガイドライン14が存在します。
ドナー色素は、マルチカラー smFRET 実験で異なるアクセプター色素にエネルギーを移す可能性があります、ので、すべての染料の相対位置は 1 つの染料だけで15,16の興奮からアクセスできません。しかし、交互にレーザー励起 (アレックス17、および18の見直し) との組み合わせでこのメソッドは、1 秒未満でサブナノ メートル分解すべての時空間的情報を提供します。
原則として、構造情報は色素間の距離を使用して実現できます高解像度はアレックスとマルチカラー smFRET 実験ではすべての蛍光強度の組み合わせから計算されます。しかし、ここで状態の識別、分離、マルチカラー smFRET が不可欠である、運動のモデルの抽出に着目します。三角形分割による構造決定「のみ」が必要な場合、高い信号対雑音比の単純な 2 色 smFRET 実験セットは実行12,19にできます。
部分的な蛍光を用いて (<img alt="Equation 1" src="/files/ftp_upload/56896/56896eq1.jpg">) 2 つの同時7間のエネルギー移動のためのプロキシとして。PFは、2 色実験の FRET 効率に類似した蛍光強度から計算されます。
<img alt="Equation 2" src="/files/ftp_upload/56896/56896eq2.jpg">
ここで、<img alt="Equation 3" src="/files/ftp_upload/56896/56896eq3v2.jpg">カラー exと励起後放出チャネルemの強度は、 cが最も長い波長を持つ受容体です。検出チャンネルは、試料室が蛍光ライトのスペクトル範囲が異なるレコードの同じ位置を表しています。励起と放射の同じ識別子はこのプロトコルで使用される (すなわち、 「青」、「緑」と"red")。
実験的欠点のため測定された蛍光強度はエネルギー転送のみならず、fluorophore およびセットアップ プロパティにも依存します。2 フルオロ間真のエネルギー伝達効率を得るために修正する測定強度があります。次の手順は、参照9に基づいています。明らかな漏れ (lk、すなわち、別の染料のため指定されたチャネルの蛍光体からの光子検出) の明白なガンマ補正係数 (ag、すなわち色素の蛍光量子収率とチャネルの検出効率) 表示するイベントを漂白アクセプター分子トレースから取得されます。
すべての可能な受容体チャネルにドナー色素の漏出、アクセプター色素漂白しますが、ドナーはまだ蛍光記録された蛍光トレース内のすべてのデータ ポイントから計算されます (<img alt="Equation 4" src="/files/ftp_upload/56896/56896eq4.jpg">)。
<img alt="Equation 5" src="/files/ftp_upload/56896/56896eq5.jpg">
漏れヒストグラムの中央値は明らかな漏洩ファクターとして使用されます。漏れの訂正後、明白なガンマ係数はトレースの同じセットによって決定されます。それはアクセプター色素の漂白時にドナーのチャネルで蛍光の変化による受容体チャネルの蛍光の変化を割ることによって計算されます。
<img alt="Equation 6" src="/files/ftp_upload/56896/56896eq6.jpg">
場所c再び、最長波長を持つ受容体の検出チャンネルです。結果の分布の中央値は、明らかな補正係数として使用されます。
各チャンネルで修正された強度は、によって得られます。
<img alt="Equation 7" src="/files/ftp_upload/56896/56896eq7.jpg">
PFによると計算されます。
<img alt="Equation 8" src="/files/ftp_upload/56896/56896eq8.jpg">
別の集団は、 PFs 多次元空間で分離できます。位置と各状態の幅は、継ぎ手の多次元ガウス関数とデータによって決まります。すべてPFトレースに基づく 1 つのグローバルな HMM の後続の最適化は、観測された挙動の定量的な説明を提供します。率の小さな変更は検出です。
Hmm は、騒々しい時間トレースの収集から状態モデルを推定する方法を提供します。システムは、任意の時点と実際の観測 (すなわち、排出量) で隠された状態はこの隠し状態20の確率関数、離散のセットのいずれかにすると見なされます。観察 smFRET データの場合、放出確率bは私の状態は私あたりは連続ガウス確率密度関数でモデル化できます。定期的に間隔をあけられた離散時間ポイントでから 1 つ別の状態への遷移、時不変であり現在の状態にだけ依存する遷移確率によると発生します。遷移行列Aでは、これらの遷移確率ijをすべて非表示のステート間を含まれています。初期状態分布<img alt="Equation 9" src="/files/ftp_upload/56896/56896eq9.jpg">状態固有の確率を与える<img alt="Equation 10" src="/files/ftp_upload/56896/56896eq10.jpg">時間トレースの最初の時間の点。最大尤度アプローチを使用して、これらのパラメーターは最高前後バウム ウェルチ アルゴリズム20,21とデータを記述するために最適化できます。最尤推定 (MLE) が得られます。最後に、最も可能性の高い観察の軌道を生成状態シーケンスはビタビ アルゴリズムで推測できます。HMM、単なる「平滑化」のデータが必要とせずデータ セットから抽出運動の状態モデルのドウェル時間を嵌合するための smFRET データ24,25,26の他の HMM 解析と対照をなして使わないヒストグラム27。HMM 解析は、Igor Pro を使用してスクリプトを内部で行われます。コードの実装は、参照21に基づいています。我々 は次のセクション 5 および 6 はこのプロトコル (https://www.singlemolecule.uni-freiburg.de/software/3d-fret) のために当社ホームページのソフトウェア キットと模範的なデータを提供します。完全なソフトウェアは、リクエストを承ります。
時間でPFを使用してデータ ポイント <-1 またはPF > 任意の検出チャンネルで 2 がすべての州 (10-200) の発生が少ない確率を割り当てられます。これは、これらのデータ ポイントで人工の遷移を防ぎます。
放出確率のパラメーターは、手順 5.7 ガウス関数による 3 D PFヒストグラムのフィットから取得されます。これらのパラメーターは、HMM の最適化中に固定されます。
提示されたアプローチで初期状態分布ベクトルと遷移行列が、トレースの全体のアンサンブルを記述するグローバルに使用されます。参照27に従って、データ セットからのすべてのN分子に基づいて更新されます。
初期状態の配布の開始パラメーターはPFヒストグラム (ステップ 5.3) の投影から決定され、遷移確率に設定されて 0.05 確率を除いて同じ状態に滞在するそのような物選ばれました。特定の状態のままにする確率を団結を正規化します。
プロファイル尤は、すべて移行率21,22不確実性の意味のある見積もりとなるための信頼区間 (Ci) を与えるためです。特定のレートの CI の境界を計算するための関心の遷移確率は尤以外の値に固定されています。これにより、テスト モデル λ'。尤度の尤度比 (LR) 検定<img alt="Equation 16" src="/files/ftp_upload/56896/56896eq16.jpg">によると特定のデータ セット0が実行されます。
<img alt="Equation 11" src="/files/ftp_upload/56896/56896eq11.jpg">
バインド パラメーターはLR 3.841、 2xの 95% の変位値を超えたときに 95% の信頼-1 自由度22,23配布。
Hsp90 を使用してメソッドの力を発揮します。この豊富なタンパク質は、細菌と真核生物で発見され、細胞ストレス応答28の一部です。がん治療29で有望な薬剤標的であります。Hsp90 は各サブユニットの30の N 末端ドメインの 1 つのヌクレオチド結合ポケットのホモです。それは、少なくとも 2 つのグローバル異なるコンフォメーション、閉鎖の 1 つと 1 つ N ターミナル オープン、V 字構造19,31,32間の遷移を受けることができます。二量体の性質は直接 Hsp90 2 つのヌクレオチド結合部位間の相互作用の問題を発生させます。
以下では、データ集録および酵母 Hsp90 とヌクレオチドの 3色 smFRET 実験の解析の手順プロトコルを提供します。蛍光に分類されたアンプ PNP のコンホメーション依存的結合 (AMP-PNP *、非加水分解性 ATP アナログ) が分析します。記述されていたプロシージャのアプリケーション研究ヌクレオチド結合のと同時に Hsp90 のコンフォメーション変化が許認可により Hsp90 の 2 つのヌクレオチド結合ポケットの間の協同性を明らかにします。

<table border="0" cellpadding="0" cellspacing="0" width="1301">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Setup</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">vibration-damped optical table&#160;&#160;</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td style="width:167px;">RS2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 473nm LX 75mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1185052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 532nm LS 50mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1261779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 594nm LS 60mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1233470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 637nm LX 140mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1196625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">laser control unit</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1234465</td>
			<td style="width:347px;">Scientific Remote</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aspheric telescope lenses</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>d=25.4mm, f=50mm and f=100mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 473/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 532/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 594/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex4</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td>FL635-10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ594RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">570DCXR</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ491RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AOTFnC-Vis</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">&#955;/4 plate</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;">AQWP05M-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CFI Apo TIRF 100x</td>
			<td style="width:445px;">Nikon Instruments Inc, Melville, NY, USA</td>
			<td style="width:167px;"></td>
			<td>high-NA objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo focus positioner MIPOS 250 CAP</td>
			<td style="width:445px;">piezosystem jena GmbH, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td>Piezo Controller NV 40/1 CLE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo stepper</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td>PZA12</td>
			<td>PZC200-KT NanoPZ Actuator Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">achromatic aspheric lenses</td>
			<td style="width:445px;">Qioptiq Photonics GmbH &#38; Co. KG, G&#246;ttingen, Germany</td>
			<td style="width:167px;">G322-304-000</td>
			<td>d=50mm, f=200mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">adjustable optical slit</td>
			<td style="width:445px;">Owis GmbH, Staufen i. Br., Germany</td>
			<td>27.160.1212</td>
			<td>max. aperture 12 x 12 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">T 600 LPXR</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">H 560 LPXR superflat</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">HC BS R635</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">525/40 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">586/20 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">631/36 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det4</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">700/75 ET Bandpass</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">optical shutters detection</td>
			<td style="width:445px;">Vincent Associates, Rochester, NY, USA</td>
			<td style="width:167px;">Uniblitz VS25S2T0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">EMCCD iXon Ultra 897</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">digital I/O card, PCIe-6535</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">syringe pump</td>
			<td style="width:445px;">Harvard Apparatus, Holliston, MA, USA</td>
			<td style="width:167px;">PHD22/2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Flow chamber</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">quartz slides</td>
			<td style="width:445px;">G. Finkenbeiner Inc, Waltham, MA, USA</td>
			<td style="width:167px;"></td>
			<td>Spectrosil2000, h=3mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">TEGADERM film</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">1626W</td>
			<td>10 x 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">spray adhesive</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">Photo Mount 050777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">glycerol</td>
			<td style="width:445px;">Carl Zeiss AG, Oberkochen, Germany</td>
			<td style="width:167px;">Immersol G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">immersion oil</td>
			<td style="width:445px;">OLYMPUS EUROPA SE &#38; CO. KG, Hamburg, Germany</td>
			<td>IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">prism</td>
			<td style="width:445px;">Vogelsberger Quarzglastechnik GmbH, Hauzenberg, Germany</td>
			<td style="width:167px;"></td>
			<td>Suprasil1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aluminium prism holder</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>custom built</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">hollow setscrews</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>with custom drilling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Tygon S3 E-3603 tubing</td>
			<td style="width:445px;">neoLab Migge GmbH, Heidelberg, Germany</td>
			<td style="width:167px;">2-4450</td>
			<td>ACF00001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PTFE tubing</td>
			<td style="width:445px;">Bohlender GmbH, Gr&#252;nsfeld, Germany</td>
			<td style="width:167px;">S1810-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sample</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">yeast Hsp90 D61C, Q385C_biotin</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>UniProt ID P02829</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Maleimide derivatives of Atto488, Atto550</td>
			<td style="width:445px;">ATTO-TEC GmbH, Siegen, Germany</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AMP-PNP*</td>
			<td style="width:445px;">Jena Bioscience, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td style="width:347px;">&#947;-[(6-Aminohexyl)-imido]-AMP-PNP-Atto647N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Fluospheres</td>
			<td style="width:445px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:167px;">F8764</td>
			<td>amine-modified, 0.2 &#956;m, yellow-green fluorescent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Software</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Andor Solis</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td>version 4.30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">LabVIEW</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td>version 2012, 32bit; misc. hardware control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">MDS control software</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td>version 2.03a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Coherent Connection</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;"></td>
			<td>version 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Igor Pro</td>
			<td style="width:445px;">WaveMetrics Inc, Portland, OR, USA</td>
			<td style="width:167px;"></td>
			<td>version 6.37</td>
		</tr>
	</tbody>
</table>

using three-color single-molecule fret to study the correlation of protein interactions

単一分子蛍光共鳴エネルギー移動 (smFRET) 生体分子のダイナミクスを調べるため広く使用されている生物物理学的技術となっています。自分のタスクを満たすために機能サイクルの相互作用パートナーと共に行動する細胞蛋白質の多くの分子機械を持っています。延長にマルチカラー 2 色 smFRET はそれに 1 つ以上の相互作用や構造変化を同時に調査することが可能。これだけではなく smFRET 実験新しいディメンションを追加しますが、イベントのシーケンスを直接研究して固定化サンプルと全反射蛍光相関相互作用を検出するユニークな可能性を提供しています顕微鏡 (TIRFM)。したがって、マルチカラーの smFRET は以前達成不可能の詳細で定量的な方法で生体分子複合体を研究するための多目的なツールです。
ここでは、蛋白質のマルチカラー smFRET 実験の特別な課題を克服する方法を示します。データを取得するため、運動情報を抽出するための詳細なプロトコルを提案します。これは、トレース選択基準、国家の分離、および 3 D アンサンブル隠されたマルコフ モデル (HMM) を用いたノイズの多いデータから状態軌道の回復が含まれています。他の方法と比較して、運動情報はドエル時間ヒストグラムから、直接 HMM からを回復しません。最尤フレームワーク キネティック モデルを批判的に評価して料金の意味のある不確実性を提供することが出来ます。
熱ショックタンパク質 90 (Hsp90) に本手法を適用しておりますはヌクレオチド結合および蛋白質のコンフォメーション変化が世界を解きほぐすことができます。これにより、Hsp90 二量体の 2 つのヌクレオチド結合ポケットの間の協同性を直接観察できます。

マルチカラー smFRET 測定は、2 つ以上の異なる相互作用部位との相関の直接検出を許可します。これは蛋白質の複合体などの多成分システムを検討するユニークな技術をレンダリングします。具体例として 3 色 smFRET 実験がここでは、プレゼンテーションに着目します。
図 1にメソッドの一般的なワー...

Watch this Scientific Journal Video about Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions at JoVE.com

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

3色 1 分子 FRET を使用して蛋白質の相互作用の相関関係を研究するには

ここでは、3色 smFRET データと 3 D アンサンブル隠されたマルコフ モデルとその解析を取得するためのプロトコルを提案する.このアプローチでは、科学者は複雑な蛋白質のシステム、協同性や相関相互作用などからキネティック情報を抽出できます。

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

using-three-color-single-molecule-fret-to-study-correlation-protein

Research

JoVE Journal

Biochemistry

10.0K ビュー.  University of Freiburg. ここでは、3色 smFRET データと 3 D アンサンブル隠されたマルコフ モデルとその解析を取得するためのプロトコルを提案する.このアプローチでは、科学者は複雑な蛋白質のシステム、協同性や相関相互作用などからキネティック情報を抽出できます。

ビデオ: Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

タンパク質 - タンパク質相互作用を研究するためのツールとしての蛍光異方性

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

生化学

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

ファストナノ位置決めシステムを用いた単一分子FRET実験から構造情報

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

足場リポソームを使用することにより、脂質近位タンパク質 - タンパク質相互作用を再構成します<em&gt;インビトロ</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

生体分子構造決定のための単分子レベルでの高精度FRET

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

細胞内タンパク質間相互作用を研究するビオチン化セル透過ペプチド

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

力の単一分子分光法のための蛋白質の共有結合固定

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

オープンソースsmfBoxを使用して、正確で正確な単一分子フレット測定を行う

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

翻訳調節におけるRNA-タンパク質相互作用を研究する光ピンセット

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

リボソームタンパク質合成の単一分子蛍光エネルギー転移研究

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

生細胞におけるタンパク質相互作用とタンパク質ダイナミクスを研究するための二色蛍光相互相関分光法

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...