我们非常感谢Chakrapani实验室对稿件批判性阅读和评论的现任和前任成员。 这项工作是由卫生部授予全国学院（1R01GM108921）和美国心脏协会（NCRP科学家发展赠款12SDG12070069）和SC的支持。

The authors have nothing to disclose.

EPR谱已被证明是在一个近乎完美的环境量化膜蛋白的构象变化无与伦比的结构方法。这种方法可以让我们一窥了在高分辨率结构遮蔽X射线晶体学和低温电子显微镜蛋白质动力学的分子机制。但是，要考虑这种做法可能会影响一般适用于其他系统，并要牢记沿途的潜在实验路障的技术限制，是非常重要的。我们讨论一些这些方面的下面一起排除故障推荐的策略。 其中较常见的问题，一个遇到具有广泛涉及突变扰动的低产量和蛋白质低聚稳定性差的技术。与复杂的多聚膜蛋白如离子通道时，这方面是进一步恶化。这是个erefore至关重要着手广泛突变之前，这两个生物化学方面进行优化。我们发现，有毒GLIC突变体的表达在BL21细胞的C41和C43菌株耐受性更好3,36另外，降低感应后温度到15日- 18ºC，降低IPTG浓度至100μM，并且包括5％甘油诱导期间似乎帮助减缓蛋白表达率和减少错误折叠的程度大概降低蛋白质聚集。在某些位置（特别是在渗透途径）半胱氨酸突变已显示出增加的信道的组成性活性和这些突变体很可能在表达造成更大的障碍。二价孔隙阻滞剂使用（10 - 25毫巴2+）诱导时减轻毒性，提高的细胞生长57这些策略已在许多情况下，在增强产量，降低蛋白质聚集和增强寡聚证明是有效的。MERIC稳定。36,58 在SDSL另一个关键步骤是标号处理的效率。而表面露出半胱氨酸应构成在MTSL反应性（&gt; 90％标记率）没有问题，其中该半胱氨酸侧链都埋和不可访问的位置，可能需要更高的自旋标记的浓度和更长的温育时间。自旋标签浓度提高至30倍摩尔过量和孵育O / N在4ºC有助于提高标记效率。此外，它也是重要的测量标记的Cys-少模板的光谱，以确定背景的贡献。对于被标记的不良部位（&lt;10％），背景信号淹没的整体光谱。在确定自旋标记效率，请注意，精度由若干包括样品体积，样品毛细管直径，谐振器内的毛细管的定位，模腔温度，和谐振器Q值的影响值（它是在能量存储在其中的能量的比例消散出来）。必须小心以维持样品和标准之间相同这些条件。此外，应该指出的是，对于不动的场所，其中所述光谱是本质宽，标记测量精度进一步下降。另外，飞行 - 电喷雾质谱法（LCT-ESI-MS）的液相色谱时间可以用来直接确定分子质量，因此贴标效率。然而，该MS灵敏度在洗涤剂和用于紧紧折叠膜蛋白破坏样品。 一旦突变体作为同源性的群体进行纯化，它以确定制剂的功能是重要的。在通道的关键区域自旋标记的突变体的膜片钳测量将揭示扰动对配体的亲和力和通道门控的效果。另外，信道特性可以在黑色L进行研究IPID膜（BLM）59或如果它发现该半胱氨酸取代，并在一定的位置上的自旋标签改变的配体灵敏度或门控动力学注射蛋白脂质体进入卵母细胞和测量由双电极电压钳59-61电流。渠道，实验条件（配体浓度，调制器，脂类成分），可以适当改变正在调查，以稳定构象状态。此外，它是要注意，EPR测量稳态条件下进行，因此，改变了信道的快速动力学的可能性较小影响的结构的解释。 显然，这种技术的主要优点是研究中所定义组合物的膜信道动态，并直接探测的动力学脂质介导的效应如何改变通道功能的能力。然而，一个限制是，一些脂质组合物可能会引起通道o的横向聚集n中的膜，因此需要脂质的适宜性来确定。45对于做引起聚合的条件下，降低蛋白质与脂质比，在较低的温度下进行重建，并缩短的重建时间的持续时间中保持通道可以帮助单分散62可替换地，通道可以在纳米圆盘，它是由两亲膜的支架蛋白（载脂蛋白A1）63的环形包围通过优化通道蛋白，脂类成分和膜支架蛋白的摩尔比的纳米脂质组件重构，它能够实现纳入个别纳米圆盘单信道单元的均匀和单分散的人口。该系统已在一些额外的优势纳米圆盘是光学透明的，并提供容易进入细胞内和细胞膜的外两侧。 最后，在水平在EPR测量，人们可能会认为是不能直接解释面临CW-EPR谱，尤其对多组分显示光谱位置。这样的构象异构性可以由在给定的蛋白质构象缓慢交换蛋白和/或自旋标记的多个方向的构象发生。确定两个场景的相对贡献是不平凡的，并且可能需要使用替代自旋标记的衍生物，或改变实验温度，64压力，65场强/微波频率，66或渗透质扰动。67 另外，在脂质体鹿测量可以通过许多因素，包括灵敏度较低，从增强分子间的相互作用导致更高的背景的贡献，并在可测量的距离范围（&lt;50埃）的减小受到限制。在一般情况下，对于较长的距离（&lt;50埃），有很大的在距离分布的宽度，由于偶极进化时间不够呃不确定性。然而，使用Q带（34千兆赫）的微波频率和标签与双功能自旋标记（具有降低的固有动态）已经显示出减轻这些局限性。54重构的纳米圆盘也已显示通过向极大改善鹿灵敏度减少从分子偶极相互作用产生的背景贡献。15 尽管有这些限制，SDSL / EPR研究，特别是在与少数天然半胱氨酸系统，已被证明是非常丰富。未来的技术进步正在向适应这个强大的方法来研究更复杂的人类渠道，其中原生变异半胱氨酸是不可行的面向。在这方面，基于68的非天然氨基酸位点特异性掺入，或SYNT备用标记策略，如那些发展标签hesis被瞄准往其他氨基酸，69似乎具有很大的希望。

在过去的十年，五聚配体门控离子通道的结构理解（pLGIC）增长的跨越式发展，由于家庭的几名成员的高分辨率结构的众人。导致在该领域的当前进展的关键因素包括，原核pLGIC渠道发现，真核生物膜蛋白表达1-3主要进展，4-6和巨大的突破，结构的测定方法。7这些结构上提供了一个明确的共识pLGIC的三维结构的整体保护。然而，这似乎落后背后两大领域是这些渠道制剂的功能特性和通道功能的机械描述。 选通的构象变化是复杂的，发生在沿着通道的长度的60埃的距离和这些转换由广泛调制膜脂。特别是，负脂质，胆固醇和磷脂已经显示调节pLGIC 8-11的功能。尽管通道功能，这些脂质成分的确切作用尚不清楚，门完整的分子机制，需要在他们自然环境研究这些通道。定点自旋标记（SDSL）和电子顺磁共振（EPR）谱是首选在复原系统研究蛋白质动力学的技术。 EPR谱不被分子大小的限制（这是NMR）或样品的光学特性（如为荧光光谱法），由此允许在天然脂质条件重构全长构建体的测量。该技术是非常敏感的，并具有相对低的样品的要求（在微微摩尔的范围）。这两个方面使该技术非常适合于大型研究膜蛋白难以超过毫克来表达数量。 利用与定点自旋标记组合EPR谱是由韦恩哈贝尔和同事开发，并已被改编为研究的范围内的蛋白质的类型。12-24 EPR数据的已被用于研究的二级结构，在该蛋白质改变构象，膜 - 插入深度，以及蛋白质 - 蛋白质/蛋白质 - 配体相互作用。 该方法涉及在由定点诱变感兴趣位置半胱氨酸取代。为确保位点特异性标记，就必须以替代天然半胱氨酸与另一个氨基酸（ 例如 ，丝氨酸）来创建一个半胱氨酸少模板。到目前为止，最流行的自旋标记是一个特定硫醇MTSL:（1-氧基-2,2,5,5-四甲基Δ3吡咯啉-3-甲基）methanethiosulfonate通过二硫键桥连接到蛋白质上。由于其高特异性，相对小的尺寸（小于色氨酸稍大），和灵活接头区域的相容性，这自旋标记已被证明具有优异的反应性，即使埋入半胱氨酸。此外，为了最大限度地提高反应性，该蛋白质的标记反应是在洗涤剂溶解的形式进行。通过尺寸排阻色谱的过量的游离自旋标记的分离后，将蛋白质重组到脂质体或限定的脂质组合物的双层模仿系统。在一般情况下，半胱氨酸诱变耐受良好的蛋白质的大部分地区，和自旋探头的相对小的尺寸会导致极小扰动的二级和三级结构。以确保该变形保留野生型功能，标记和重构通道可以通过膜片钳测量来研究。 然后将标记的官能蛋白进行光谱测量，其基本上提供三种主要类型的信息:由linesh 12,14,15,20,22,23,25-27自旋探针动力学猿分析;探头顺松弛剂无障碍;和距离的分布。27 EPR距离通过两种不同的方法进行测定。第一种是基于所述连续波（CW）技术，其中，从自旋标签之间偶极相互作用产生的频谱展宽（在8 - 20埃的距离范围内）。用于确定距离28,29的第二个是一个脉冲的EPR方法，其中，距离测量可以延长至70埃。30-34在双电子电子共振（鹿），在自旋回波振幅振荡被分析以确定的距离和距离的分布。这里自旋回波是在偶极相互作用的频率调制。总之，这些参数被用来确定蛋白质的拓扑结构，二级结构元件，和蛋白质的构象变化。

<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.

自旋标记GLIC突变体的生化特征 按照上述描述的方案通常产生GLIC-MBP融合蛋白在10〜 - 12毫克/升培养物。虽然这个值可以在不同的突变体而变化，特别是对于埋在蛋白质内的位置时，产率可能会显著损害。在这些情况下，培养体积可能需要扩大。融合的裂解构建由HRV3C蛋白酶进行O / N方便。通过运行在SDS PAGE凝胶上的样品中，我们发现，这种切割是30内完成 - 60分钟。 （ 图1A）。大小排阻凝胶过滤柱纯化，GLIC主要洗脱与11.3毫升保留体积五聚体。 MBP的级分洗脱以15ml和聚集在柱（〜7.0mL）中的空隙体积中洗脱。自旋标记正值洗脱结束时（〜24ml）中（ 图1B） 基于FRET检测在各种膜脂监测GLIC复溶的聚集 FRET的测定用于在重构囊泡评估GLIC五聚体的聚合（距离&lt;50埃的膜中来在一起）。 图2示出了来自GLIC重量数据（与C27，标记或荧光素或四甲基罗丹明），并于E重构.coli极性脂质，POPC:POPG（3:1），POPE:POPG（3:1）或asolectin。荧光测量表明，在GLIC重组asolectin没有表现出FRET，甚至冻结/解冻样品（称为促进凝集的条件）所产生的FRET 43水平变化很小。出于这个原因，asolectin是选择用于这些STU使用的功能和光谱测量脂质混合物死亡。 在脂蛋白GLIC的宏观行为 在重组蛋白脂质体纯化GLIC的功能特性的特点是膜片钳测量。从重量GLIC从响应于的pH跳的内侧出补丁代表宏观当前记录示于图3在-100毫伏保持电位，切换至pH3.0缓冲产生迅速激活向内的电流。（10 - 100毫秒），该衰变到秒43内的峰值幅度的约10％（1 - 3秒， 图3）。电流从这个宏观腐烂或脱敏切换回中性pH恢复。而在里面出补丁通道是在浴pH值的变化敏感，有在吸管溶液无pH变化的影响（数据未显示），表明GLIC被在一个严重主要取向ction使得在补丁中的胞外结构域所面临的浴/溶液热交换器。 结构重排在通道激活 在RT，自旋探头可以采用多个旋转异构构象，和谱线形状不仅是自旋标签侧链相对于肽骨架的运动，也给骨干波动和全球蛋白运动敏感。因此，在EPR lineshapes变化可以被证明是监测蛋白的构象变化的优良的诊断工具。 图4示出了光谱在位置L241（17）中的R1孔隙衬M2区域的胞外疏水区域（位置突出如黑圆图4， 插图 ）在pH 7.0和pH 3.0。两种条件下的光谱显示的差异，无论是在信号幅度和的程度动感扩大。与功能测量42，由EPR信号报告的构象变化也完全可逆的。频谱展宽由探头的动生约束和由偶极耦合的影响两者。以确定两个，L241（17）的贡献是共同标记有抗磁自旋的标签。下标记的和充分标记的光谱在pH 7.0和pH 3.0（ 图4B）进行了比较。偶极增宽在该位置的范围内降低时，通道被下标，它表示不同的亚基之间自旋标签之间的相互作用。 图5示出用于在M4螺旋的两个位置，其位于所述通道的周边光谱。 ΔHø-1为中心的线宽度（在插图示出）的逆测量。作为氮氧化物的旋转运动被减少，因为形成期间目睹的叔或季接触，线路宽度增加（并因此ΔHö-1减少）对于任何特定动生的几何形状。与此相反，从而增加在移动的探头的自由结构运动反映为○-1增加ΔH。 F312R1是更多的移动而R296R1是不动的，与他们的接触和高度受限的环境相一致，分别为。 基于所述辅助参数，在蛋白上的特定位置处的自旋标记物可以被分类为以下之一:埋蛋白质核无法进入水或脂质内，暴露于水性溶液的表面上，或者在表面暴露于脂质（ 图5和6）。对于一系列连续的部位的所获得的可访问参数然后可以被用来确定一个区域的二级结构，探针蛋白质的区域接触，测量蛋白质的倾斜膜内，估计界面环的浸入深度，以及跨膜螺旋内识别水前庭和脂质结合口袋26作为一个例子，在图6中显示，F312R1具有电饱和曲线对于O 2的相对高磷1/2，相比于R296R1，暗示侧链在位置F312是脂露出。此外，F312R1也是比较R296R1其在脂质 - 水界面的位置一致，以NiEDDA更容易获得。另一方面，L180R1在细胞外结构域的环C区是移动的，并向NiEDDA，符合其在暴露于水的循环位置。 探针动力学和辅助数据在给定的位置提供关于整个蛋白质倍之内的特定位置局部结构信息。对于人确定这些参数沿着该蛋白质的残基的inear序列可以使用基于傅立叶分析方法变换方法作进一步分析。这些分析是评估在EPR环境参数的周期性变化，并在预测和定向的二级结构，引导有益的。对于特定蛋白质片段，离散傅立叶变换被计算为在该段相邻位置（ω）之间的角度的函数的功率谱P（ω）。然后该曲线被用来确定频谱的主要角频率分量相比，对于α螺旋的预期（ω）的值（80 - 120º）或β链（150 - 180º）。 <img alt="公式3" src="/files/ftp_upload/54127/54127eq3.jpg" /> 其中，P（&#39;ω&#39;）的傅里叶变换的功率谱作为角频率ω的函数，n是在该段中的残基的数目，是在该位置的环境参数，P（"ω"）获取的价值评估ω= <img alt="公式6" src="/files/ftp_upload/54127/54127eq6.jpg" />最大化P（&#39;ω&#39;）。 另外，周期性指数参数（给出了预测的二级结构的显着性）被计算作为区域的为表示该光谱的总面积的一个给定的结构元件的角功率谱下的比率。然后，滑动窗口方法是用来识别的开始和一个给定的二级结构元件的端部。相对方向相对于该蛋白的其余部分这一部分可以从P（&#39;ω&#39;）的时刻的方向进行估算。 对于α螺旋: <img alt="公式4" src="/files/ftp_upload/54127/54127eq4.jpg" /><img alt="公式5" src="/files/ftp_upload/54127/54127eq5.jpg" />这些方法已经成功应用于多个系统来确定三维拓扑结构，并预测蛋白质功能14,23,24,41,44-53潜在的构象变化。 从EPR确定分布的距离 跨亚氮氧化物的距离可以从电子 - 电子偶极相互作用来确定。在CW-EPR， 如图4，通过空间偶极相互作用导致的频谱变宽和是标签之间的接近的功能。距离可达约15 - 20 A，拓宽（比较充分标光谱和下标记谱之间）的程度可以用于半定量地估计使用距离傅立叶去卷积方法。28,29 间亚基距离（&lt;50埃）可以使用双电子-电子共振（鹿）方法测量。30-34鹿数据被收集洗涤剂样品中或在因为在脂质体54这些测量相关的限制的纳米圆盘重构。用于洗涤剂中的缓冲液A自旋标记的样品（〜100μM）用0.5mM DDM，30％（重量/体积）甘油被用于冷冻保护。偶极时间演化数据使用标准鹿四脉冲协议（π/ 2）83ķ得到MW1-τ1-（π）MW1-τ1-（π）MW2-τ2-（π）MW1-τ2回波55在脉冲EPR谱仪在Q波段频率（33.9 GHz）的工作。为（π/ 2）MW1和（π）MW1的脉冲长度是10和20毫微秒，分别与40纳秒为（π）MW2。鹿信号然后背景校正假设一个三维均匀backgroUND并分析了吉洪诺夫正规化31,56来确定距离平均距离和分布。 用于通过鹿实验在pH 7.0确定在M4（F314R1）的代表性位置的分子内距离被示出在图7中由于有在GLIC五聚体在五个旋标签，至少两个距离预计。对应一到相邻亚基和不相邻的亚基另一方面，虽然额外的峰值可能从备用旋转异构自旋方向出现。鹿信号衰减和相应的概率分布列。第一短距离峰（〜42A）表示相邻亚基之间的距离，和第二峰（〜53A）对应于非相邻的距离。为对角线距离的峰是在短距离，这是可能的，因为超出60埃可靠的测量结果被低估不是实验条件下可行由于与背景校正技术困难。 <img alt="图1" src="/files/ftp_upload/54127/54127fig1.jpg" /> 图1. 生化特征自旋标记GLIC突变体 。自旋标记-GLIC的MBP标签的蛋白水解切割后（A）的代表性的凝胶过滤色谱图。该峰对应于GLIC五聚体和游离的MBP。 （B）表示从凝胶过滤汇集的完整无缺的单体GLIC-MBP融合蛋白（凝胶过滤前）和MBP和GLIC分数SDS-PAGE。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54127/54127fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54127/54127fig2.jpg" /> 图2. FR<html> ET基于测定监测GLIC复溶的聚集状态中的各种膜脂。FRET示为样品测量后O / N复溶（左）和冷冻/解冻的样品（右侧）之后。 （从原来的图修改）。43 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54127/54127fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54127/54127fig3.jpg" /> 图3. 在GLIC的脂蛋白宏观行为。在从重组asolectin泡切除一个由内而外的补丁，GLIC受pH值激活使用快速的解决方案交换跳跃。的信道被看作激活〜10毫秒和1的时间常数脱敏 - 3秒。 （从原来的图修改）。43ig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54127/54127fig4.jpg" /> 图4 结构重排期间信道激活。（A）的代表在孔的位置处衬M2螺旋，响应于pH变化显示在幅度和线的形状的变化的CW-EPR谱。在每一种情况下，光谱被标准化为自旋的总数。自旋标记位置被示为黑色圆圈。仅两个亚单位示为清楚。 （B）EPRspectra显示了由自旋相互作用偶极增宽。在虚线的谱从下标记通道（在抗磁性标签的存在），并在实线的来自完全标记的信道。插图显示振幅归一化的光谱的重叠。充分标记的条件下扩大高箭头亮起。扫描宽度为150 G.（从原来的图修改）。36 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54127/54127fig4large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="如图5" src="/files/ftp_upload/54127/54127fig5.jpg" /> 图5. 残留的具体环境参数由功率饱和进行测量。在跨膜结构域的M4螺旋两种截然不同的立场谱宽（ΔH0）和溶剂可（P 1/2）的测量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54127/54127fig5large.jpg" target="_blank">请点击此处查看大图这一数字。</a> <img alt="图6" src="/files/ftp_upload/54127/54127fig6.jpg" /> 图6 的环境<html>一个代表性的循环C残基在GLIC的胞外区。自旋标准化CW-光谱和相应的辅助功能的三围L180R1。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54127/54127fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54127/54127fig7.jpg" /> 脉冲-EPR途径（鹿）。GLIC结构表示Phe314的位置和相应的相邻和非相邻亚基Cβ-Cβ距离图7 的距离测量 。 CW-光谱这个位置显示在右侧。背景减去DEER-回波强度暗算演化时间，并使用无模型吉洪诺夫正则洗涤剂样品适合。相应的自旋间距离分布（ 右 ）。PLOAD / 54127 / 54127fig7large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

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.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

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 次观看.  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.

视频: 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).

小型低聚物形成膜蛋白的沉降平衡:组氨酸质子对五聚体稳定性

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

科研

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.

表面修饰碳纳米管通过土柱运输

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.

弥漫分散剂/粒子绑定机制反射红外光谱鉴定功能性油墨

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.

利用循环伏安法、紫外-近红外光谱和 EPR 光谱电化学对有机化合物进行分析

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.

35-吡啶N-氧化物脱水的合成、结晶和光谱表征

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.

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

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.

局部钙离子梯度对大泡囊膜重塑的响应

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.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

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.

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

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.

信息承载Peptoids及其序列定向动态共价自组装的合成

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