私たちは、彼女のデータの使用のために北京呉に感謝。この作品は、JEKにNSFのキャリア賞によってサポートされていました

1。膜タンパク質のフォールディングのための〜50 nmの直径小さな単層小胞（SUV車）の調製<ol><li> 1,2 -ジミリストイル- sn -グリセロ-3 -ホスホコリン（DMPC）、クロロホルム中脂質の溶液を保存用バイアルの量あたり20 mgで購入し、清浄なガラスバイアルに小分けされています。窒素ガスの層は、脂質の酸化を防ぐために、各バイアルに添加され、そしてバイアルをキャップし、パラフィルムで密封されている。バイアルは使用するまで-20℃の冷凍庫に保存されます。 </li><li>クロロホルムの20 mgの脂質のアリコートを含む単一のバイアルは、各実験に使用されます。バイアルの内容は、無溶剤のままになるまで1時間窒素ガスの流れを用いて乾燥している。 </li><li>乾燥脂質は、〜30秒間、水浴の超音波処理を用いて20 mMリン酸カリウム緩衝液（pH = 7.3）1mLに再懸濁する。中断された脂質のこの溶液は濁り表示され、円錐形の底部にプラスチックを15 mLの試験管に移している。リン酸緩衝液の別の1mLのアリコートはもともと脂質が含まれている空のガラス製バイアルに添加され、そして超音波処理が繰り返されます。溶液は、次いで同じ15 mLの試験管に追加されます。チューブの最終容量は、この株式の小胞の溶液中で5 mg / mLの脂質の濃度で、その結果、4 mLのようになるまでこのプロセスを4回の合計が繰り返されます。 </li><li>脂質ベシクル溶液4 mLのボリュームが置かれている暖かい（〜30℃）の水浴、および50％のデューティサイクルで1時間ultrasonicatorのマイクロチップを使用して超音波処理。水浴の目的は2つあります。最初に、それがために超音波処理プロセスのあまりに高温になることから、脂質溶液を防ぎます。 DMPCのために、この転移温度は〜23℃であり、第二に、温かいお風呂は、水性脂質溶液の温度は超音波処理中に、二重層の相転移温度以上に保つことができます</li><li>超音波処理ソリューションは、超音波処理器の先端からゴミを削除するには、0.22μmのシリンジフィルターを通過し、このろ液は37℃で一晩平衡化される℃です。 </li></ol> 2。初期蛍光アンフォールディングカーブのための試料調製<ol><li> 20mMリン酸緩衝液（pH7.3）で10 Mの尿素の原液が行われます。それは尿素の大量の溶液中でかなりのボリュームを占有しているので、水が固体（およびその逆）に追加されることが不可欠です。この尿素溶液はきれい、空の瓶に溶質を追加し、目的の最終容量まで水を加えることによって行うことができます。このソリューションは、水浴や溶質を溶解するために、攪拌しながらホットプレート上で温めて必要となる場合があります。尿素は吸湿性であるため、尿素の原液の実際の濃度は、屈折率を用いて決定してください1。 </li><li> 20mMリン酸液（pH7.3）の株式の緩衝液が行われます。 </li><li>折り畳まタンパク質のストック溶液を調製した。この株式のタンパク質溶液は、8 M尿素、20 mMリン酸緩衝液で〜200μMの蛋白質の濃度を持つ必要があります。 </li><li>蛍光研究のためのサンプルは、次の方法で製造されています。株式の蛋白質の適切な量（2.3節）、株式の脂質溶液（5 mg / mLの脂質、第1項）、株式10 Mの尿素溶液（セクション2.1）、および株式のリン酸緩衝液（セクション2.2）〜4を含む試料を作るために結合されてμMタンパク質、1 mg / mLの脂質、および1 Mの単位で0〜8 M尿素。各サンプルの合計量は200μLです。例のスプレッドシート（​​表1）はリストのボリュームが200μMストックタンパク質溶液を使用して必要なことが含まれています。 </li><li>ブランクサンプルは2.4節で説明するように作られている、しかし、タンパク質が追加されません。他の成分の濃度は、2.4節の場合と同じになるように、タンパク質の代わりに、8 M尿素溶液4μLを加えてもよい。これらのブランクが散乱し、タンパク質の蛍光スペクトルに現れる他のバックグラウンド信号を減算するために使用されます。 </li><li>セクション2.4および2.5からのサンプルは完全な折り畳みと平衡を確保するための蛍光スペクトルの測定の前に少なくとも2時間、37℃でインキュベートされている。 </li></ol> 3。蛍光スペクトルの測定各サンプルとブランクのトリプトファンの蛍光は、定常状態蛍光光度計を用いて測定されます。蛍光スペクトルは、チロシン残基の励起を避けるために、290nmの励起波長で記録、および305〜500nmのスキャンする必要があります。典型的な入口と出口のバンドは3 nmである。波長の増加と積分時間は、信号対雑音比を最適化することができます。波長の増加と積分時間の典型的な値は1nm /ステップと、それぞれ0.5秒/ステップ、です。膜は、温度が二重層の相転移温度以上である場合にのみ合成脂質に一般的に倍のタンパク質。したがって、DMPCのこのケースでは、試料の温度は30℃一定に保たれています℃に160μLの容量を持つ微量石英キュベットは、これらの元で利用されているperiments。 4。アンフォールディング膜タンパク質のギブス自由エネルギーの初期アンフォールディング曲線と推定の世代<ol><li> XYデータセットの形で蛍光スペクトルは、イゴールプロ、Matlabの、起源、またはExcelなどのソフトウェアにロードされます。 </li><li>尿素と脂質小胞のバックグラウンドからの信号は、次の手順を使用して減算する必要があります。 脂質小胞と尿素の特定の濃度の存在下でタンパク質の=生の蛍光スペクトルスペクトル。 ない蛋白質は、ブランク試料に存在しない、脂質小胞とスペクトルと同じ尿素の濃度を含む、対応するブランク試料のスペクトルは、B =生の蛍光スペクトル。 補正スペクトル=スペクトラムA - C *スペクトルB. Cは1.0に典型的に等しいスカラです。いくつかのケースでは、Cが原因で発生する散乱問題のより小さいか1.0より大きいオーバーまたは〜305〜320 nm領域の近減算下。 </li><li>最大蛍光の波長、λmaxは 、それぞれの尿素濃度の補正されたスペクトルから表にあらわしています。折り畳まれたタンパク質のそれは〜330 nmの間に完全に展開した膜タンパク質の典型的な値は350 nmである。表形式の波長は折り畳ま人口の割合にはλmaxの値を相関させるため0.0から1.0の範囲にはλmaxの値（〜350 nmまで〜330）の範囲を変換することで展開した分数に変換されます。例えば、330 nmのλmaxの値が0.0に対応し、350nmのは、1.0に対応し、340nmのは、展開した分数の面で0.5に対応しています。折り畳まタンパク質のこの画分を尿素の濃​​度に対してプロットされている。前述したように、尿素の濃度は、屈折率を測定することによって実験的に決定されるべきである。 </li><li>我々は、タンパク質が2つの状態のいずれかに存在することができることを前提としています。折り曲げたり広げた。分数のプロットは、fを伸ばしたのに対し、尿素の濃度、Cは 、式に適合することができます：2 <img src="/files/ftp_upload/2669/2669eq2.jpg" alt="式（2）" /> 係数mは、変性剤濃度に対する自由エネルギーの変化率に相当し、そしてC mは折られた人口が展開人口が等しくなる中間点の尿素の濃度に相当する。これらの値は、Rは （0.001987 kcal / molの•K）気体の法則は一定である。データ解析ソフトウェアによって最小二乗フィッティングから得られたフィッティングの変数であり、Tは （303 K）の温度である。 </li><li>近似​​から得られる係数は、尿素の存在下で展開のギブスの自由エネルギー量（kcal / mol）を決定するために利用されています： <img src="/files/ftp_upload/2669/2669eq3.jpg" alt="式（3）"/> 。 </li></ol> 5。アンフォールディングのギブス自由エネルギーのより正確な測定のためのアンフォールディング曲線を最適化。 <ol><li>上記の展開曲線はλmaxで最大の変化を引き起こす尿素濃度の範囲を明らかにする。この範囲は一般的に小さくなり、展開の大半は、この小さな範囲内で発生します。展開の自由エネルギーを正確に決定するために、追加のサンプルが最適化された最小二乗フィッティングのために必要な多くのデータポイントが含まれているプロットを取得するために、この領域で分析されます。より正確に展開曲線のこの重要な領域の形状を明らかにするために0.2 Mの単位で4 Mの尿素 - タンパク質が2と4 M尿素との間で繰り広げられるたとえば、サンプルは2を含むことを行うことができます。それは、それぞれの尿素濃度でブランクのスペクトルを測定する必要はなく、すべての〜0.5 Mのステップ空白のスペクトルを測定するのに十分です。 </li><li>このプロットから得られた展開の自由エネルギーは、オリジナルの展開曲線で得られたものとは多少異なるが、より正確な値を反映している可能性があります。 </li></ol> 6。代表的な結果： <table border="1"><tbody><tr><td>サンプル</td><td>最後の[尿素]、M </td><td>株式のタンパク質（μL） </td><td>株式の脂質（μL） </td><td>株式10 M尿素（μL） </td><td>株式バッファー（μL） </td></tr><tr><td> 1 </td><td> 〜0.16 </td><td> 4 </td><td> 40 </td><td> 0 </td><td> 156 </td></tr><tr><td> 2 </td><td> 1 </td><td> 4 </td><td> 40 </td><td> 20 </td><td> 136 </td></tr><tr><td> 3 </td><td> 2 </td><td> 4 </td><td> 40 </td><td> 40 </td><td> 116 </td></tr><tr><td> 4 </td><td> 3 </td><td> 4 </td><td> 40 </td><td> 60 </td><td> 96 </td></tr><tr><td> 5 </td><td> 4 </td><td> 4 </td><td> 40 </td><td> 80 </td><td> 76 </td></tr><tr><td> 6 </td><td> 5 </td><td> 4 </td><td> 40 </td><td> 100 </td><td> 56 </td></tr><tr><td> 7 </td><td> 6 </td><td> 4 </td><td> 40 </td><td> 120 </td><td> 36 </td></tr><tr><td> 8 </td><td> 7 </td><td> 4 </td><td> 40 </td><td> 140 </td><td> 16 </td></tr></tbody></table> 表1。蛍光サンプルを作るのに必要な原液の量<img src="/files/ftp_upload/2669/2669fig1.jpg" alt="図1" /> 図1単一のトリプトファン残基を含んでいる〜5μMの代表的な膜蛋白質のトリプトファン蛍光スペクトル。蛋白質の（A）生の蛍光スペクトル（スペクトル4.2から）、空白の（B）生の蛍光スペクトルは、（4.2からスペクトルB）、（C）4.2からスペクトルを修正しました。 <img src="/files/ftp_upload/2669/2669fig2.jpg" alt="図2" /> 図2は、。多数の尿素の濃度については、図1から膜タンパク質のトリプトファン蛍光スペクトルを修正しました。 <img src="/files/ftp_upload/2669/2669fig3.jpg" alt="図3" /> 図3。4.4に式に合わせて、図2のデータから得られた曲線を広げる。ギブスの自由エネルギーは、セクション4.5に従って計算されます。

<ol>
	<li>Shirley, B. A., Shirley, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Folding and Stability. , 177-190 (1995).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+and+Analysis+of+Urea+and+Guanidine+Hydrochloride+Denaturation+Curves.">Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves.</a> Methods Enzymol. 131, 266-279 (1986).</li><li>Lau, F. W., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Assessing+the+Stability+of+a+Membrane+Protein.">A Method for Assessing the Stability of a Membrane Protein.</a> Biochemistry. 36, 5884-5892 (1997).</li><li>Burgess, N. K., Dao, T. P., Stanley, A. M., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Barrel+Proteins+that+Reside+in+the+Escherichia+coli+Outer+Membrane+in+Vivo+Demonstrate+Varied+Folding+Behavior+in+Vitro.">&#946;-Barrel Proteins that Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro.</a> J. Biol. Chem. 283, 26748-26758 (2008).</li><li>Booth, P. J., Curnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+scene+investigation:+membrane+proteins.">Folding scene investigation: membrane proteins.</a> Curr. Opin. Struct. Biol. 19, 8-13 (2009).</li><li>Hong, H., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+coupling+of+integral+membrane+protein+stability+to+lipid+bilayer+forces.">Elastic coupling of integral membrane protein stability to lipid bilayer forces.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 4065-4070 (2004).</li><li>Sanchez, K. M., Gable, J. E., Schlamadinger, D. E., Kim, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Tryptophan+Microenvironment,+Soluble+Domain,+and+Vesicle+Size+on+the+Thermodynamics+of+Membrane+Protein+Folding:+Lessons+from+the+Transmembrane+Protein+OmpA.">Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA.</a> Biochemistry. 47, 12844-12852 (2008).</li></ol>

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現在のプロトコルは、トリプトファン残基を含む膜関連タンパク質およびペプチドの曲線を展開の生成を説明します。ここでは、トリプトファンの蛍光がタンパク質が折り畳まと合成脂質小胞に挿入、または溶液中で折り畳まれているかどうかを反映することを前提としています。追加のような変性剤濃度との自由エネルギーの二状態フォールディングと線形依存性などの仮定、、現在のレ?...

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thermodynamics of membrane protein folding measured by fluorescence spectroscopy

膜タンパク質の折り畳みは、基本的な健康関連の両方の意味を持つ新たなトピックです。細胞の基礎となってタンパク質のこのユビキタス家族の折りたたみの包括的な研究の必要性の膜タンパク質の豊富さ。さらに、ミスフォールドタンパク質に関連する疾患を特徴付けるために我々の能力の進歩は、タンパク質の折り畳みの分野で重要な実験と理論の努力を動機としている。この重要な分野で急速な進歩は、残念なことに、膜タンパク質や折りたたみ機構の複雑さに関連する固有の課題によって妨げられている。ここでは、Eからの代表的な内在性膜タンパク質を、変性剤、ΔG · H 2 Oの存在下で展開のギブス自由エネルギーの熱力学特性を測定するための実験手順の概要を説明大腸菌 。このプロトコルは、変性剤濃度の関数としての折り返しと展開した状態の平衡個体数を決定するために蛍光分光法の応用に焦点を当てています。合成脂質小胞だけでなく、データ解析の手順の重要なステップの準備のための実験的な考慮事項が強調表示されています。この手法は汎用性があり、温度やpH、同様の脂質とミセルの様々な折りたたみ環境などの変性剤の様々なタイプ、と追求されることがあります。現在のプロトコルは、以下に説明する一連の基準を満たす任意の膜または可溶性タンパク質に一般化することができるものです。

Watch this Scientific Journal Video about Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy at JoVE.com

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

トリプトファン蛍光法による膜タンパク質の折り畳みのギブス自由エネルギーを得るためにこのビデオの記事の詳細は、実験手順。

蛍光分光法により測定膜タンパク質フォールディングの熱力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

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18.2K ビュー.  University of California San Diego - UCSD. トリプトファン蛍光法による膜タンパク質の折り畳みのギブス自由エネルギーを得るためにこのビデオの記事の詳細は、実験手順。

ビデオ: Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

マイクロ流体液滴のプラットフォームのための蛍光検出法

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

生物工学

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

タンパク質の折り畳みを研究するためのマイクロ流体ミキサー

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：膜タンパク質のタンパク質 - タンパク質相互作用を識別するために、生化学的ツール<em&gt;インビボ</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

細胞内小胞の追跡への応用：変形二光子顕微鏡での3d軌道の追跡

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

ブリルアン分光法のための細胞外マトリックスのタンパク質繊維の調製

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

非正規のアミノ酸の選択圧による 'ゴールデン' 蛍光と質量分析法と蛍光タンパク質解析工学

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

ナノ製剤中のエンドトキシン検出カブトガニ リムラス ライセート (ラル) 試金を使用して

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

自律性小胞成長のためのペプチド膜前駆体のベシキュロ合成

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

生体膜力プローブ分光法による分子ばね定数解析

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

蛍光タンパク質中のプロリン類似体によるプロリンの残基特異的交換:骨格の「分子手術」が折り畳みと安定性にどのように影響するか

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...