著者たちは、オーストラリアシンクロトロンのMX2ビームラインでの支援のために精製微結晶、Daniel ErikssonとTom Caradoc-Daviesの写真を提供するChan-Sien Layと、Monash UniversityのFlowCore施設からのKathryn FlanaganとAndrew Fryga貴重な援助。

<ol>
	<li>Tari, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+structural+biology+in+drug+discovery.">The utility of structural biology in drug discovery.</a> Methods Mol Bio (Clifton, N.J). 841, 1-27 (2012).</li><li>Gati, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+crystallography+on+in+vivo+grown+microcrystals+using+synchrotron+radiation.">Serial crystallography on in vivo grown microcrystals using synchrotron radiation.</a> IUCrJ. 1 (2), (2014).</li><li>Redecke, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natively+inhibited+Trypanosoma+brucei+cathepsin+B+structure+determined+by+using+an+X-ray+laser.">Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.</a> Science. 339 (2013), 227-230 (2013).</li><li>Shi, D., Nannenga, B. L., Iadanza, M. G., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+electron+crystallography+of+protein+microcrystals.">Three-dimensional electron crystallography of protein microcrystals.</a> eLife. 2, e01345 (2013).</li><li>Evans, G., Axford, D., Waterman, D., Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+microcrystallography.">Macromolecular microcrystallography.</a> Crystallog. Rev. 17 (2), 105-142 (2011).</li><li>Smith, J. L., Fischetti, R. F., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-crystallography+comes+of+age.">Micro-crystallography comes of age.</a> Curr Opin Struct Biol. 22 (5), 602-612 (2012).</li><li>Boudes, M., Garriga, D., Coulibaly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+the+Many+Facets+of+Protein+Microcrystallography.">Reflections on the Many Facets of Protein Microcrystallography.</a> Aust J Chem. 67 (12), 1793-1806 (2014).</li><li>Nelson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+cross-beta+spine+of+amyloid-like+fibrils.">Structure of the cross-beta spine of amyloid-like fibrils.</a> Nature. 435 (7043), 773-778 (2005).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+organization+of+cypovirus+polyhedra.">The molecular organization of cypovirus polyhedra.</a> Nature. 446 (7131), 97-101 (2007).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atomic+structure+of+baculovirus+polyhedra+reveals+the+independent+emergence+of+infectious+crystals+in+DNA+and+RNA+viruses.">The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.</a> Proc Natl Acad Sci U S A. 106 (52), 22205-22210 (2009).</li><li>Johansson, L. 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著者は何も開示することはない。

このプロトコルは、過去に見過ごされていた非常に小さな結晶の分析を容易にする目的で、微結晶を分析する2つのアプローチを提供する。 微結晶の浄化のための重要なステップ 提示されたプロトコールは、モデル系としてSf9細胞で発現されたボンビクス・モリ CPV1ポリヘドリンを用いて最適化されている。しかしながら、 in vivo微結晶は、機械的耐性に大きなばらつきを示す。例えば、昆虫細胞中で増殖したカテプシンBの針状結晶は、剛性であり、機械的ストレスに対して高度に耐性であり、本明細書に記載されたものと同様のプロトコールを用いて精製することができる14 。一方、ホタルルシフェラーゼ結晶は、培養された昆虫細胞で同様の針状形態で増殖し、すぐに細胞溶解に溶解する38 。したがって、 インビボの抽出および精製のためのプロトコール結晶は、ステップ1.3,2a.3-4,2b.4,3.1.3-4および3.2a.5に記載されている特定のケースについて試行錯誤的に適合させる必要がある。 壊れやすい結晶の場合、細胞破片からの（部分的な）分離は、低速遠心分離（ すなわち 200×g）または反復超音波処理の代わりにチューブの底部にスクロースクッションを加えることによって達成され得る。一般的な規則として、回折データ収集目的のために調製された結晶サンプルは高度に精製される必要はない。従って、結晶が腐敗する前に低温冷却することが迅速な精製が好ましい。 インビトロ および インビボ 微結晶 への応用 両方のプロトコールは、ここではインビボで成長した結晶の分析で説明されているが、この方法は、精製された組換えタンパク質からの結晶トレイ中で増殖した微結晶に容易に使用することができる。それでi）メッシュ支持体は、ピペットで移すのではなく、結晶滴から微結晶を直接回収するために使用することができる。 ii）結晶の量が制限要因となる可能性があるので、過剰な液体をメッシュ支持体から吸い取って結晶の過剰な除去を避けるために特別な注意を払う必要がある。 iii）従来の結晶学（文献30参照）で行われているように、異なる凍結保護緩衝液を試験する必要がある。 一方、 生体内で成長した結晶では、セルロのアプローチが強く推奨されます。細胞は精製された結晶よりも視覚化および操作が容易であるため、このアプローチは、ビーム時間の使用に関してより効率的であり、微結晶化の経験がほとんどまたはまったくない研究者にとってよりアクセスしやすくなります。それはまた、抽出およびpurの影響を受け得る脆弱な結晶に、より適している<html>化学的環境の変化、周囲のタンパク質の希釈および機械的損傷による細胞からの感染。さらに、細胞は結晶に保護環境を提供し、凍結保護物質の添加の必要性を緩和する。凍結保護処理の省略は、以前に欠陥の蓄積を防止することが示されており、室温でのデータ収集のために、個々の結晶間の同形性を改善した40 。 イン・セルロ・プロトコルはまた、凍結保護溶液中でのインキュベーションの必要条件をバイパスし、極低温でのデータ収集も可能にする。全体として、 セルロデータの収集には2つの主な利点があります.1）ビームタイム20の一定期間、より良い品質と高解像度のデータを生成することができます。 2）細胞環境において結晶化したタンパク質を維持し、生物学的に関連するリガンドを保持する機会を増やす。 ontent &quot;&gt; 技術の限界 連続微結晶学の本質的な複雑さは、データ収集中に生成される重要な数の部分データセットである。結果として、データ処理はますます時間を要するプロセスとなる。ただし、データ処理の基本的なワークフローは大部分が自動化できます。例えば、同形結晶の最適な選択は、ブレンド36に実装され、例えば、階層的クラスター分析28によって決定することができます。さらに、X線自由電子レーザー（XFEL）の解析のために開発されたプログラムは、シンクロトロンビームライン2で収集されたシリアル微結晶学データを処理するために適合させることができ、メッシュおよび結晶検出の自動ラスタリングは、データ収集2、41。

生物学的高分子の高分解能構造の決定のためのX線結晶学の使用は、過去20年間にわたり安定した進行を経験している。非専門家による研究者によるX線結晶学の益々の取り込みは、ライフサイエンスの多くの分野におけるこのアプローチの民主化を実証している1 。 歴史的に、約10μm以下の寸法を有する結晶は、構造決定のために使用できないとしても、困難であると考えられてきた。世界的なシンクロトロン放射源の専用マイクロフォーカスビームラインの利用可能性と、微結晶を操作するためのツールの開発などの技術的進歩により、X線マイクロ結晶の幅広い利用を妨げてきた多くの障壁が取り除かれました。シリアルX線microcrystallography 2,3及びマイクロ電子回折4ヘクタールの進歩構造決意用マイクロおよびナノ結晶の使用が可能でなく、大きな結晶5、6、7の使用に時々好ましくないだけであることが示されまし。 これらの進歩は、第一のペプチド8及び昆虫ウイルス9、10によって生成される天然の結晶の研究に適用しました。それらは現在、膜タンパク質や大きな複合体などの最も難しい系を含む多様な生物学的巨大分子に使用されています11 。これらの微結晶の分析を容易にするために、それらはメソ 、特に膜タンパク質12およびマイクロ流体チップ13において分析されている。 これらの新規な微結晶化方法の利用可能性は、古典的なインビトロ crystallogenesisに代わるものを提供する構造生物学14、15、16のための新たな経路として結晶体内。残念なことに、 インビボ結晶が産生されても、細胞からの精製中のリガンドの分解または喪失、シンクロトロンビームラインでの結晶の操作および視覚化の困難さ、退屈なX線回折実験などのいくつかの障害が残る。別の結晶はまた、任意の精製工程17、18、19せずに細胞内で直接分析されているように。比較分析はcelluloアプローチにおけるように精製された結晶およびより高い解像度20の収率データの分析よりも効率的であることを示唆しています。 このプロトコルはタンパク質微結晶学の新しい研究者を支援する傾向があった。それは、シンクロトロンビームラインでのX線回折実験のための試料調製および操作に焦点を当てた方法論を提供する。 2つの選択肢が、古典的な微結晶学のための単離された結晶またはフローサイトメトリーによってソーティングされた結晶含有細胞を用いて、セルロ分析で提案されている（ 図1 ）。

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf9 cells</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SF900-SFM insect medium</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1L cell culture flask</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Shaking incubator for insect cell culture</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mL conical tubes</td>
			<td style="width:127px;">Falcon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge with swing buckets for 50mL tubes</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sonicator equiped with a 19mm probe</td>
			<td>MSE Soniprep 150&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass slides</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Influx cell sorter&#160;</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hampton Heavy atom screens</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Micromesh</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paper wick</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">MX2 microfocus beamline</td>
			<td style="width:127px;">Australian Synchrotron</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
			doi:10.1071/CH14455.</td>
		</tr>
	</tbody>
</table>

microcrystallography of protein crystals and in cellulo diffraction

多くのシンクロトロン施設で高品質のマイクロフォーカスビームラインが出現したことにより、最大の次元で10μm以下の結晶の日常的な解析が可能になりました。本発明者らは、X線結晶学によるタンパク質微結晶の構造決定のための2つの代替のワークフローを提示し、特にin vivoで成長した結晶に焦点を当てる。微結晶は、超音波処理によって細胞から抽出され、微分遠心分離によって精製されるか、または結晶含有細胞のフローサイトメトリーによる細胞選別の後、セルロで分析される。場合によっては、精製された結晶または結晶含有セルを、実験段階的な調整のために重原子溶液に浸漬する。これらの試料は、マイクロメッシュ支持体上に塗布し、液体窒素中でフラッシュ冷却することにより同様の方法で回折実験用に調製する。我々は、単離された微結晶および結晶質結晶の連続回折実験を簡単に説明し、モデル構築および精密化に適したデータセットを生成するためにマイクロフォーカスシンクロトロンビームラインを使用している。 これらのワークフローは、昆虫細胞に組換えバキュロウイルスを感染させることによって産生されるBombyx mori cypovirus 1（BmCPV1）ポリヘドリンの結晶で例示される。このケーススタディでは、セルロ分析では精製された結晶の分析よりも効率的であり、発現から精製まで約8日間で構造が得られます。

生体内微結晶を用いた構造決定のための代替方法の両方の概要が示されている（ 図1 ）。多面体は、超音波処理および遠心分離によって容易に精製することができる。密度のため、ピペットで取り除くことができる破片の層の下のチューブの底に層を形成する（ 図3aおよび3b ）。次いで、サンプルを数回の超音波処理およびペレットの白色および白亜面から判断されるように十分なレベルの純度に達するまで洗浄する（ 図3c ）。 イン・セルロ・アプローチでは、非感染細胞のフローサイトメトリー・プロファイルを結晶含有細胞のプロファイルと比較し、どの細胞集団を選択して結晶含有細胞を他の細胞（ 図4 ）。この例では、多面体を含む細胞は、非感染細胞よりも高い側方散乱を有する。散乱パターンの他の違いが他の結晶型で観察され、それに応じてゲーティングを修正する必要があります。 精製された微結晶または結晶含有細胞をマイクロメッシュ上にピペットで移す（ 図5 ）。トリプタンブルーで染色された細胞は、ほとんどのシンクロトロンビームライン（ 図5b ）で利用可能なカメラを使用して簡単に視覚化することができますが、精製されたクリスタルは小型であるためX線ビームを識別し整列させるのに手間がかかります（ 図5c ）。データを収集するときは、センタリング手順を最小限に抑え、同一の細胞または水晶を2回曝露しないようにするか、または何も欠損させることを避けるために、グリッドパターンで進めることが最善です（ 図5a ）。データ収集者回折限界の違い、放射線損傷の影響、同型異性の可能性の欠如を考慮して注意深く処理する必要があります。 <img alt="図1" class="xfigimg" src="/files/ftp_upload/55793/55793fig1.jpg" /> 図1 ： 生体内微結晶を用いた構造決定のための2つの方法の概要。微結晶は、組換え発現系を用いて目的のタンパク質を過剰発現させることにより、細胞培養において産生される。特定の場合には、微結晶はまた、特定の条件下で細胞によって自然に産生され得る。一番上の行は、細胞が溶解され、結晶が分画遠心分離によって精製される古典的アプローチを示す。精製された微結晶をマイクロメッシュ上にピペットで移す。過剰の液体を除去した後、凍結保護剤溶液を添加し、過剰の液体を再び吸い取り、メッシュをフラッシュ冷却する。 X線回折の場合<html>結晶をX線ビームと慎重に整列させ、これはデータ収集における律速段階である可能性がある。一番下の行は、 セルロのアプローチです。細胞をフローサイトメトリーで選別して、結晶含有細胞を特異的に選択する。視覚化を容易にし、マイクロメッシュ上に広げるために細胞をトリパンブルーで染色する。この場合、凍結保護剤の添加は任意である。 X線回折実験では、センタリングプロセスを容易にするシンクロトロンビームラインで利用可能な典型的なカメラを使用して、細胞を容易に視覚化します。 スウェーデン、ウプサラのGE Healthcare ABによる自動液体クロマトグラフィーシステムのイメージ<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig1large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a> <img alt="図2" class="xfigimg" src="/files/ftp_upload/55793/55793fig2.jpg" /> = &quot;xfig&quot;&gt;図2 ： 生体内微結晶の例。インビボ結晶の選択された例：（ a ）サイフォウイルス多面体を有するSf9細胞; （ b ）エンテロポックスウイルススフェロイドを有するLD652細胞; （ c ）Cry3A結晶を有するバチルス・チューリンゲンシス （矢印）; （ d ） Trypanosoma bruceiカテプシンB結晶を有するSf9細胞; （ e ）カルシニューリン結晶を有するSf21細胞（矢印）; （ f ）PKAを有するCOS7細胞単層：Inka1結晶; （ g ）IgG結晶を有するCHO細胞。許可を得て、14、18、29、37、39、42から再生されました。スケールバー=10μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig2large.jpg" target="_blank">ラージを表示するにはここをクリックしてください</a>この図の赤版です。 <img alt="表1" src="/files/ftp_upload/55793/55793table1.jpg" /> 表1： 組換えタンパク質を発現する培養細胞で増殖したインビボ結晶のまとめ。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793table1large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a>

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

タンパク質微結晶を用いたX線結晶解析のためのプロトコールが提示されている。精製後又はcelluloにおける微結晶-grown インビボで分析する2つの例が比較されます。

タンパク質結晶の微結晶化および<em&gt;セルロで</em&gt;回折

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

microcrystallography-of-protein-crystals-and-in-cellulo-diffraction

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K ビュー.  Monash University. タンパク質微結晶を用いたX線結晶解析のためのプロトコールが提示されている。精製後又はcelluloにおける微結晶-grown インビボで分析する2つの例が比較されます。 

ビデオ: Microcrystallography of Protein Crystals and In Cellulo Diffraction

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

共免疫沈降により核および細胞質画分中のタンパク質間相互作用の可視化と<em&gt;その場で</em&gt;近接ライゲーションアッセイ

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

生化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

タンパク質結晶の成長を使用して異なる寸法自動結晶化その場の動的光散乱と相まって

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 の 1: タンパク質・ タンパク質代謝物の複合体の並列解析のための親和性の浄化をワンステップ

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

測定と酸素消費量全体の解釈飛ぶ頭セグメント

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

蛍光顕微鏡を使用して競争力のある滴定による転写因子の DNA 結合親和性の高感度測定

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

タンパク質の折り畳み、トランスポート、および放射性パルス追跡による生体細胞の劣化の解析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

完全自律型の特性と生体高分子の結晶からのデータ収集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

転写におけるタンパク質とタンパク質の相互作用を研究するための生体層干渉計(BLI)の応用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.