私たちは、原稿上の重要な読書とコメントChakrapaniラボの現在および過去のメンバーに非常に感謝しています。 この作品は、国立衛生研究所の助成金（1R01GM108921）とアメリカ心臓協会（NCRPサイエンティスト開発グラント12SDG12070069）によって、およびSCにサポートされていました。

The authors have nothing to disclose.

EPR分光法はネイティブに近い環境での膜タンパク質の立体構造変化を定量化するに比類のない構造的アプローチであることが証明されました。このアプローチは、私たちにX線結晶構造解析とクライオ電子顕微鏡から高解像度の構造に隠されているタンパク質のダイナミクスの分子の詳細にのぞき見することができます。しかし、他のシステムへの一般的な適用に影響を与える可能性があり、また、心の中で道に沿って潜在的な実験的な障害を保つために、このアプローチの技術的な制限を考慮することが重要です。私たちは、以下のトラブルシューティングのための推奨戦略に沿って、これらの態様のいくつかを議論します。 1は、大規模な突然変異摂動を伴う技術で遭遇した、より一般的な問題の中でタンパク質の収率が低く、貧しいオリゴマー安定性があります。このようなイオンチャネルのような複雑な多量体膜タンパク質で作業するときに、この態様はさらに悪化します。それは番目です広範囲の突然変異誘発に着手する前に、これらの2の生化学的な側面を最適化することが重要erefore。私たちは、有毒なGLIC変異体の発現がBL21細胞のC41とC43株における忍容性であることがわかった3,36また、15に誘導後の温度を低下させる- 。、18ºC100μMのIPTG濃度の減少、および5％グリセロールを含みます誘導中のタンパク質発現の速度を遅くし、ミスフォールディングの程度を減少させることによって、おそらくタンパク質の凝集を低下させることに役立つと思われます。 （特に、透過経路における）特定の位置にシステイン突然変異はチャネルの構成的活性を増加させることが示されており、これらの変異体は、発現に大きな障害をもたらす可能性があります。二価の細孔遮断薬の使用-誘導時（10 25ミリモルのBa 2+）、毒性を軽減し、細胞増殖を向上57これらの戦略は、歩留まりを向上させるタンパク質凝集を低下させ、オリゴ向上に多くの場合に有効であることが証明されています。量体安定性。36,58 SDSLにおける別の重要なステップは、ラベリング処理の効率です。表面に露出したシステインがMTSL反応性（&gt; 90％ラベリング利回り）で問題ないべきであるが、システイン側鎖が埋設され、アクセスできません位置は、より高いスピンラベルの濃度およびより長いインキュベーション時間を必要とするかもしれません。 30倍モル過剰のスピンラベルの濃度を増加させ、4ºCでO / Nでインキュベートして標識効率を向上させることに役立ちます。また、バッ​​クグラウンドの寄与を決定するために、標識されたシステインのないテンプレートのスペクトルを測定することも重要です。不十分なラベル付けされているサイト（&lt;10％）の場合は、バックグラウンドシグナルは、全体的なスペクトルを圧倒する。スピン標識効率を決定しながら、精度がサンプル容積、試料毛細管の直径は、共振器内の毛細管の位置決め、キャビティ温度、及び共振器Qを含む多くの要因によって影響されることに注意してください値（アウト散逸エネルギーを介してその中に蓄積されたエネルギーの比率です）。ケアは、サンプルと標準の間で同一これらの条件を維持するために注意しなければなりません。また、スペクトルは本質的に広範である不動部位ため、標識の測定精度がさらに低下することに留意すべきです。代替的に、飛行エレクトロスプレーイオン化質量分析（LCT-ESI-MS）の液体クロマトグラフィー時間は、直接分子量を決定し、したがって、効率を標識するために使用することができます。しかし、MS感度は洗剤でしっかりと折りたたまれた膜タンパク質のためのサンプルのために危険にさらされています。 突然変異体は均質な集団として精製されると、調製物の機能性を確認することが重要です。チャネルの主要地域におけるスピン標識変異株のパッチクランプ測定は、リガンド親和性およびチャネルゲーティングに対する摂動の影響を明らかにします。また、チャネル特性はブラックLで研究することができますIPID膜（BLM）59または卵母細胞へのプロテオリポソームを注入し、2電極電圧クランプによって電流を測定することによって。59-61特定の位置でシステイン置換およびスピンラベルのリガンド感受性またはゲーティング動態を変化させることが判明した場合チャネルは、実験条件（リガンド濃度、モジュレーター、脂質組成物）は、調査中の立体配座状態を安定化させるために適切に変化させることができます。また、EPR測定は、定常状態の条件下で製造されるため、チャネルの急速な速度の変化は、構造の解釈に影響を与える可能性が低いことに留意すべきです。 明らかに、この手法の主な利点は、定義された組成物の膜内のチャネルのダイナミクスを研究するための直接ダイナミクスの脂質媒介効果は、チャネル機能を変化させる方法をプローブする能力です。しかし、制限がいくつかの脂質組成物は、チャネルoの横方向の凝集を引き起こす可能性があることですN膜とは、したがって、脂質の適合性を確認する必要がある。45原因凝集を行う条件については、タンパク質対脂質比を低下させる低温での再構成を行うこと、および再構成時間の持続時間を短縮するチャネルを維持するのに役立つことができます単分散62代わりに、チャネルは両親媒性の膜骨格タンパク質（アポリポタンパク質A1）の弁輪に囲まれたナノスケールの脂質集合であるナノディスクに再構成することができる。63チャネルタンパク質、脂質成分、および膜骨格タンパク質のモル比を最適化することにより、個々のナノディスクに組み込まれ、単一のチャネルユニットの均質かつ単分散集団を得ることができます。このシステムは、ナノディスクが光学的に透明であり、細胞内および細胞膜の細胞外両側に簡単にアクセスを提供することで、いくつかの追加の利点を有しています。 最後に、レベルでEPR測定の、1は、特に多成分のスペクトルを表示する位置について、解釈することは簡単ではありませんCW-EPRスペクトルに直面する可能性があります。このようなコンフォメーションの不均一性は、ゆっくりと所定のタンパク質の立体構造中のタンパク質および/またはスピンラベルの複数の方向の立体配座を交換から生じ得ます。 2つのシナリオの相対的な寄与を決定することは容易ではありません、代替スピンラベル誘導体、または実験温度を変え、64気圧、65電界強度/マイクロ波周波数、66またはオスモライトの摂動によっての使用を必要とするかもしれない。67 さらに、リポソーム中の鹿の測定は感度が低く、強化された分子間相互作用に起因する高いバックグラウンドの寄与、および測定可能距離範囲（&lt;50Å）の減少を含む多数の要因によって制限される場合があります。一般的に、より長い距離（&lt;50オングストローム）のために、偉大な存在であります不足による双極進化倍に距離分布の幅の小胞体の不確実性。しかし、（減少した固有ダイナミクスと）二官能性スピン標識とのQバンド（34 GHzの）マイクロ波周波数とラベリングの使用は、これらの制限の一部を緩和することが示されている。ナノディスク54再構成も大幅によってDEER感度を改善することが示されています分子間双極子相互作用から生じるバックグラウンドの寄与を減少させる。15 特に、いくつかのネイティブのシステインを有するシステムでは、これらの制限、SDSL / EPRの研究にもかかわらず、著しく有益であることが判明しています。今後の技術の進歩は、変異ネイティブシステインが実現可能ではないかもしれない、より複雑な人間のチャンネルを研究するために、この強力なアプローチを適応に向けています。この点で、部位特異的な非天然アミノ酸の組み込み、68またはSYNTに基づくものなどの代替の標識戦略の開発他のアミノ酸をターゲットにしているラベルのhesis、69は偉大な約束を保持するように見えます。

過去10年間、五量体リガンド依存性イオンチャネル（pLGIC）の構造的理解は、家族のいくつかのメンバーの高分解能構造の群衆に起因して、飛躍的に成長してきました。フィールド内の現在の進歩につながった主な要因は、構造決意アプローチ中の原核pLGICチャンネルの発見、真核生物の膜タンパク質の発現に1-3主要な進展、4-6と驚異的なブレークスルーを含む。7これらの構造は、上の明確なコンセンサスを提供しますpLGICの3次元構造の全体的な保全。しかし、背後に引きずるように見える二つの主要な分野は、これらのチャネル製剤の機能的特徴およびチャネル機能のメカニズムの説明があります。 ゲーティングコンフォメーション変化は複雑であり、チャネルの長さに沿って60オングストロームの距離にわたって起こり、これらの遷移は広くによって調節されます膜脂質。具体的には、負の脂質、コレステロール、およびリン脂質はpLGIC 8-11の機能を調節することが示されています。チャネル機能におけるこれらの脂質成分の正確な役割は不明のままであるが、ゲーティングの完全な分子的理解は、彼らのネイティブ環境でこれらのチャネルを研究が必要となります。部位特異的スピンラベリング（SDSL）および電子常磁性共鳴（EPR）分光法は、再構成されたシステムでは、タンパク質のダイナミクスを研究するための選択肢の技術です。 EPR分光法（蛍光分光法のように）分子の大きさ（NMRのように）、またはサンプルの光学的特性によって制限され、それによって天然の脂質状態に再構成全長構築物の測定が可能です。技術は非常に敏感であり、（ピコモルの範囲内で）比較的低いサンプル要件があります。これらの両方の側面がミリグラム以上で表現することが困難な大規模な膜タンパク質を研究するための技術は非常に適して作ります量。 部位特異的スピン標識と組み合わせて、EPR分光法を使用することは、ウェインハベルらによって開発された、およびタンパク質の種類の範囲を研究するために適合されている。12-24 EPRデータは、二次構造を調査するために使用されている、タンパク質の変化を立体構造、膜挿入深さ、およびタンパク質 - タンパク質/タンパク質 - リガンド相互作用。 この方法は、部位特異的突然変異誘発により、目的の位置にシステイン置換を含みます。部位特異的な標識化を確実にするために、システインレステンプレートを作成する別のアミノ酸（ 例えば 、セリン）で天然のシステインを置換することが必要です。これまでのところ、最も人気のあるスピン標識は、チオール特異的MTSLである：（1-オキシル2,2,5,5-テトラメチルΔ3-ピロリン-3-メチル）ジスルフィド結合のブリッジを介してタンパク質に付​​着methanethiosulfonate。その高い特異性、比較的小さいサイズ（トリプトファンよりわずかに大きい）、およびフレキシへリンカー領域の性は、このスピン標識も埋め込みシステインに優れた反応性を有することが示されています。さらに、反応性を最大化するために、タンパク質の標識反応は、界面活性剤可溶化形態で行われます。サイズ排除クロマトグラフィーにより過剰の遊離スピン標識を分離した後、タンパク質は、リポソームまたは定義された脂質組成物の二重層模倣システムに再構成されています。一般的には、システイン突然変異誘発はよくタンパク質のほとんどの地域では許容され、かつスピンプローブの比較的小さいサイズは、二次および三次構造に最小限の摂動を引き起こします。修飾は、野生型の機能を保持することを保証するために、標識され、再構成されたチャネルは、パッチクランプ測定によって研究することができます。 標識機能性タンパク質は、その後、本質的に情報の主に3つのタイプを提供する分光測定に供される：lineshにより12,14,15,20,22,23,25-27スピンプローブダイナミクスを猿の分析;常磁性緩和剤に対するプローブのアクセス;距離分布27 EPR距離は、2つの異なるアプローチによって測定されます。最初は（8 - 20Åの距離範囲）をスピン標識との間の双極子相互作用に起因するスペクトルの広がりは連続波（CW）技術に基づいている。距離を決定するために使用されている28,29秒パルス、EPRであります距離測定は、二重電子電子共鳴（DEER）で70Å。30-34にまで拡張することができる方法は、スピンエコー振幅の振動は、距離と距離の分布を決定するために分析されます。ここでのスピンエコーは双極子相互作用の周波数で変調されます。一緒に、これらのパラメータは、タンパク質のトポロジーは、二次構造要素、およびタンパク質コンホメーション変化を決定するために使用されます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Site-Directed Mutagenesis and Cys mutations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x PfuUltra HF reaction buffer</td>
			<td>Agilent Technologies</td>
			<td>600380-52</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPS</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>N0447L</td>
			<td>10mM each dNTP</td>
		</tr>
		<tr>
			<td>pfu Ultra DNA polymerase</td>
			<td>Agilent Technologies</td>
			<td>600380-51</td>
			<td>2.5 U/ul</td>
		</tr>
		<tr>
			<td>DPNI</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>R0176S</td>
			<td>20,000 U/ml</td>
		</tr>
		<tr>
			<td>XL10 GOLD</td>
			<td>Agilent Technologies</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC media</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>B9020S</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientfic</td>
			<td>BP905</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media</td>
			<td>Invitrogen</td>
			<td>127957084</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>C43 competent cells</td>
			<td>Lucigen</td>
			<td>60446</td>
			<td></td>
		</tr>
		<tr>
			<td>Expression and Purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientfic</td>
			<td>D16</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher Bioreagents</td>
			<td>BP1421-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Amresco</td>
			<td>J850</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Bioreagents</td>
			<td>BP229</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Amresco</td>
			<td>0705</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Amresco</td>
			<td>0781</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (isopropyl-thio-&#946;-galactoside)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C25</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma Life Science</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma Life Science</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Amresco</td>
			<td>M145</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptine</td>
			<td>Amresco</td>
			<td>J580</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Amresco</td>
			<td>J583</td>
			<td></td>
		</tr>
		<tr>
			<td>DDM (n-Docecyl-&#946;-D-Maltopyranoside)</td>
			<td>Anatrace</td>
			<td>D310S</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>E8021L</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Amresco</td>
			<td>K831</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientfic</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Acros Organics</td>
			<td>329915000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200GL</td>
			<td>GE Healthcare</td>
			<td>17-5175-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Empty polypropylene Chromatography column</td>
			<td>BioRad</td>
			<td>731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-Directed Spin Labeling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) Methanethiosulfonate</td>
			<td>Toronto Reaserch chemicals Inc</td>
			<td>O873900</td>
			<td></td>
		</tr>
		<tr>
			<td>(1-acetoxy-2,2,5,5-tetramethyl-&#916;3-pyrroline-3-methyl) methanethiosulfonate</td>
			<td>Toronto Reaserch chemicals Inc</td>
			<td>A167900</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>J.T. Baker</td>
			<td>9224-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Reconstitution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Asolectin lipid</td>
			<td>Avanti polar lipids Inc</td>
			<td>541602C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biobeads (Polystyrine beads)</td>
			<td>Bio Rad</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher chemicals</td>
			<td>A413</td>
			<td></td>
		</tr>
		<tr>
			<td>FRET</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein-maleimide</td>
			<td>ThermoFisher Scientific</td>
			<td>F-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine-maleimide</td>
			<td>ThermoFisher Scientific</td>
			<td>T-6027</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC</td>
			<td>Avanti polar lipids Inc</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>POPG</td>
			<td>Avanti polar lipids Inc</td>
			<td>840457C</td>
			<td></td>
		</tr>
		<tr>
			<td>E.Coli polar lipid extract</td>
			<td>Avanti polar lipids Inc</td>
			<td>100600C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Life Science</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>EPR measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TPX plastic capillaries</td>
			<td>Bruker</td>
			<td>ER221</td>
			<td></td>
		</tr>
		<tr>
			<td>EDDA (Ethylenediamine-N, N&#39;-diacetic acid)</td>
			<td>Aldrich</td>
			<td>158186</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni(OH)2</td>
			<td>Aldrich</td>
			<td>283622</td>
			<td></td>
		</tr>
	</tbody>
</table>

site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

サイト監督スピン標識および五量体リガンド依存性イオンチャネルのEPR分光研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

site-directed-spin-labeling-epr-spectroscopic-studies-pentameric

Nanyang Technological University

Research

JoVE Journal

Chemistry

10.6K ビュー.  Case Western Reserve University. This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

ビデオ: Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

小オリゴマー形成膜タンパク質の沈降平衡：五量体安定性に対するヒスチジンプロトン化の影響

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

化学

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

土壌カラムを通して表面修飾カーボンナノチューブの輸送

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.

Formulation of stable, functional inks is critical to expanding the applications of additive manufacturing. In turn, knowledge of the mechanisms of dispersant/particle bonding is required for effective ink formulation. Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) is presented as a simple, inexpensive way to gain insight into these mechanisms.

機能性インク中の分散剤/粒子​​結合機構の反射赤外分光識別を拡散

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

サイクリックボルタンメトリー、紫外-可視-近赤外、および EPR 分光電気化学を使用して有機性化合物を分析するには

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the methodology for non-substituted pyridines in 1957 in a 12 h process, but no X-ray suitable crystals were obtained. The substituted ring used in the methodology presented here clearly influenced the addition of water molecules into the asymmetric unit, which confers a different nucleophilic strength in 1. The X-ray suitable crystal compound 1 was possible due to the stabilization of the negative charge in the oxygen by the presence of two water molecules where the hydrogen atoms donate positive charge into the ring; such water molecules serve well to construct a supramolecular interaction. The hydrated molecules may be possible for the alkaline system that is reached by adjusting the pH to 10. Importantly, the double methyl substituted ring and a reaction time of 5 h, makes it a more versatile method and with wider chemical applications for future ring insertions.

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

Herein, we report the synthesis and crystallization of 3,5-lutidine N-oxide dehydrate by a simple protocol that differs from the classical synthesis of pyridine N-oxide. This protocol utilizes different starting material and involves less reaction time to yield a new solvated supramolecular structure, which crystallizes under slow evaporation.

合成、結晶化、および 3, 5-ルチジンNの分光学的キャラクタリゼーション-酸化物の脱水

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

安定した二環性 Aziridinium イオンと開環の Azaheterocycles の合成のための準備

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

ローカライズされたカルシウム イオン勾配に対応した膜小胞の膜を改造

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

合成と化反応のサイト選択的に作成するためのチオール反応性の試薬の変更 Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

金属イオン認識の構造とメカニズムを決定するイオン移動質量分析技術と金属結合オリゴペプチドの酸化還元活性

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

情報を持つペプトイドの合成と、その配列指向動的共有自己集合

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...