这项工作由 PDOC (13-0017-01 至钲和 ANR-14-CE09-0020-01 至 a.1)、"未来投资" 方案达思波尔多/CNRS (pep 2016 至钲) 供资, ANR-10-IDEX-03-02 钲, 研究, 基金会倾吐 la Médicale () 的资助 (FRM-AJE20140630090 到 a.1), FP7 计划 (FP7-PEOPLE-2013-CIG 到 a.1) 和欧洲研究理事会 (紧急救济委员会) 根据欧洲联盟的地平线2020研究和创新计划 (紧急救济协调员开始授予 a.1, 协议 639020) 和项目 "WEAKINTERACT。

作者没有什么可透露的。

固态核磁共振 (SSNMR) 是一种在原子水平上表征大分子蛋白组件的方法。SSNMR-based 结构确定的核心问题之一是被调查系统的频谱质量, 它允许建立各种精度的3D 结构模型, 通常包括低分辨率模型 (包含次结构元素和小3D 信息) 到 pseudo-atomic 3D 结构。从多维 SSNMR 实验中提取的结构信息的数量和质量是计算装配的高分辨率核磁共振结构的关键。
所描述的协议依赖于检测13c-13c 和15N13c 结构限制, 要求记录多个 2D (有时 3D) 高信噪比的频谱。在适度 MAS 频率 (和 #60; 25 赫), 样品被引入到转子的大小为 3.2-4 毫米直径允许蛋白质数量高达〜50毫克, 依赖于样品水合。转子内的样品量与 SSNMR 谱中的信噪比成正比, 这是检测远距离约束的决定性因素, 是其明确的赋值。
在序列共振分配和约束集合中, 谱分辨率是一个关键参数。为了获得最佳的结果, 需要优化样品制备参数, 特别是在纯化亚基和组装条件 (pH, 缓冲, 震动, 温度,等)。对于样本优化, 建议为已观察到的几个不同的条件准备未标记的样本, 并在每个准备好的样本上记录 1D 1H-13C CP 频谱 (在步骤2.1 中描述)。光谱用于比较光谱决议和分散在不同的准备, 根据哪些最佳的条件可以被确定。
SSNMR 数据的质量在很大程度上取决于核磁共振采集参数的选择, 特别是极化转移步骤。高磁场强度 (≥600 MHz 1H 频率) 的使用对于高灵敏度和光谱分辨率是非常重要的, 在面对复杂目标 (如大分子蛋白组件) 时需要。
在许多情况下的限制因素是光谱仪的可用性。因此, 在光谱仪会议之前, 明智地选择要准备的样品。无论如何, 一个统一的13C、 15N 标记的示例是执行顺序和 intra-residual 共振分配的先决条件。为 solid-state 核磁共振技术分配的蛋白质参见71。在中等 MAS 频率下的高分子组件的结构测定需要有选择的13C 标记的样本;用于检测远程13c-13c 和13c-15N 联系人示例基于 13-13c 和 2-13c-gylcerol 和/或 1-13c-和 2-13c-葡萄糖标记是通常使用, 如上所述。两种标记方案之间的选择是基于频谱信噪比和分辨率。为了区分内部和分子间的长程接触, 混合标记和稀释样品已显示有效。
简而言之, 原子 SSNMR 结构研究的关键步骤是: (i) 需要优化亚基和组件的制备, 以获得优异的样品数量和质量, (ii) 光谱仪场强度和采集参数必须慎重选择;(三) 3D 结构确定需要有选择性的标记策略, 所需数据的数量取决于数据质量和补充数据的可用性。
尽管它适用于范围广泛的超分子系统, 从膜蛋白到 homomultimeric 纳米物质, 但 SSNMR 通常受限于镁量的 isotopically 标记材料的需要。最近的技术发展在超快 MAS (小时赫) SSNMR 开辟了大道到1H 检测到的核磁共振, 并将最小样本量的极限推到 sub-mg 72,73,74。然而, 对于详细的结构研究, 13C 标记的样本是必不可少的, 这限制了 SSNMR 的应用, 以样本组装的体外或系统中所表达的生物生存在最小的培养基上的细胞SSNMR 是一种新兴的方法 (用于审阅, 请参阅75,76,77,78)。
在 SSNMR 应用中获得高分辨率3D 结构的一个重要因素是光谱分辨率: 组件内的固有构象异质性可以限制光谱分辨率和频谱分析。剩余的特定13C 标记在某些情况下可能提供一种替代方法, 以获取有关战略残滓的特定距离信息, 以便获得结构模型 (最近的示例见79,80)。
SSNMR 为3D 结构决心仍然要求收集几个数据集以经常长的数据采集时间在老练仪器, 取决于方法和系统几天到星期在600-1000 兆赫 (1H 频率)光谱仪.因此, 在 in-depth SSNMR 的研究中, 获得光谱仪的时间可能是一个限制因素。
在 homomultimeric 蛋白组件的情况下, 导致 SSNMR 数据足够的质量, 以确定大量的结构限制, 如在3,57,64,70, SSNMR仍然无法进入微观维度。因此, 在de 从头SSNMR 结构确定 homomultimeric 组件、EM 或每长度 (MPL) 数据时, 理想地补充 SSNMR 数据以导出对称参数。SSNMR 数据仅提供原子和分子间的界面
SSNMR 是高度互补的结构技术, 如 EM 或 MPL 测量, 但数据也可以完美地结合的原子结构获得 X 射线结晶学或解决核磁共振的突变或截断亚基。越来越多的研究可以在文献中发现, 不同结构数据的结合允许确定大分子组件的原子3D 模型 (参见图 6中的典型例子)。
在结构生物学领域, SSNMR 作为一种有前途的研究方法应运而生。原子级的不溶和晶的组件,即提供原子尺度的结构数据。在这方面, SSNMR 是为分子组装的溶液核磁共振和 X 射线结晶学的垂饰, 包括其原生环境中的膜蛋白和蛋白质组合, 如病毒包络、细菌丝或 amyloids, 以及 RNA 和RNA 蛋白质复合体 (参见例如81)。它的高度多才多艺的应用在体外和在细胞的上下文, 例如跟踪次要, 第三和第四纪结构变动, 辨认相互作用表面与伙伴分子在原子尺度 (例如82) 和映射分子动力学在装配的复合体的上下文, 表明 SSNMR 的重要潜力在未来结构研究复杂生物分子汇编。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组件</td>
			<td>M9 培养基</td>
		</tr>
		<tr>
			<td>nacl</td>
			<td>0.5 克/升</td>
		</tr>
		<tr>
			<td>2PO4</td>
			<td>3克/升</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>6.7 克/升</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>1毫米</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>10μ m</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>1μ m</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>100μ m</td>
		</tr>
		<tr>
			<td>记忆维生素混合100X</td>
			<td>10毫升/升</td>
		</tr>
		<tr>
			<td>13C-葡萄糖</td>
			<td>2克/升</td>
		</tr>
		<tr>
			<td>15NH4Cl</td>
			<td>1克/升</td>
		</tr>
	</tbody>
</table>
表 1:重组蛋白最低表达培养基的组成生产于 大肠杆菌 BL21 单元格.

魔术角纺 solid-state 的研究进展核磁共振光谱学 (SSNMR) 为大分子蛋白质组件的结构表征提供了一个原子分辨率的有效工具。这些蛋白质组件是无处不在的系统, 在许多生物过程中扮演着重要的角色。他们的分子结构、相互作用和动力学由 SSNMR 研究是可接近的, 如已经显示为病毒 (衣1) 和细菌感染机制 (分泌系统2,3, 毛4), 膜蛋白质复合物5,6,7,8和功能 amyloids 9,10,11。这种类型的分子组装也可以引发病理, 如在神经退行性疾病的蛋白质聚集在错误, 淀粉样状态和导致异常细胞行为或细胞死亡12,13。蛋白质组合通常是由蛋白质亚基的倍数拷贝的对称齐聚物形成的, 包括纤维、细丝、孔、管或纳米颗粒等各种形状的超分子。第四级体系结构是由蛋白质亚基之间的弱相互作用来组织空间和时间组装, 并允许复杂的生物功能。在这些组件的原子尺度上进行结构上的调查是对高分辨率技术的挑战, 因为它们的内在不, 而且常常是它们的 non-crystallinity 限制了常规 X 射线晶体学或溶液核磁共振的使用方法.魔术角纺 (MAS) SSNMR 是一种新兴的技术, 获得原子分辨率数据的不溶性高分子组件, 并已证明它的效率, 以解决3D 原子模型的越来越多的复杂生物分子系统, 包括细菌丝, 淀粉样蛋白组件和病毒颗粒14,15,16,17,18,19,20, 21,22。在高磁场、方法学发展和样品制备方面的技术进步, 已经建立了 MAS SSNMR, 成为一种健壮的方法, 在各种环境中研究不溶性蛋白质, 特别是在其生物相关大分子组装状态或细胞膜, 使该技术的高度互补的低温电子显微镜。在许多情况下, 这样的蛋白质组合具有很高的对称性。MAS SSNMR 利用这一特性, 因为 homomolecular 组件中的所有蛋白质亚基都具有相同的局部结构, 因此几乎相同的 SSNMR 签名, 大大降低了分析的复杂性。
一种有效的协议结构研究大分子蛋白组件由适度 MAS (和 #60; 25 赫) SSNMR 介绍了在这个视频, 并可以细分为不同的步骤 (图 1)。我们将展示 SSNMR 结构研究的工作流程的关键阶段, 例如丝状蛋白组件 (参见图 1中突出显示的步骤), 除蛋白质亚基纯化外, 不同的蛋白质大会, 但对结构研究至关重要, 而不进入 SSNMR 光谱学的技术/方法细节, 并在网上提供专门教程的结构计算。虽然本议定书将主要集中在 MAS 条件下进行的 solid-state 核磁共振实验, 使用对齐的生物环境23,24,25,26,27, 如对齐的 bicelles, 允许在无 MAS 技术的情况下研究蛋白质构象和动态蛋白质-蛋白质相互作用。我们将显示蛋白质的表达和组装步骤, 以及关键的 SSNMR 谱的记录和他们的分析和解释。我们的目标是提供对结构分析管道的洞察力, 使读者能够通过 SSNMR 技术对大分子组装进行原子解析结构研究。
该协议包含3部分:
1. 固态核磁共振样品生产
作为一个 solid-state 核磁共振分析的先决条件, 大分子组装的蛋白质组分需要表达, 同位素标记, 纯化和组装体外到本机一样的复杂状态 (例如, 请参见图 2).为确保高核磁共振灵敏度, 在13c 和15n 标记中的同位素富集需要通过使用最小细菌表达介质, 并辅以13c 和15n 源, 如一致的13分别标有葡萄糖/甘油和15NH4Cl。在议定书的后期阶段, 有选择地使用13c 标记的示例生成的带有选择性13c 标记的源 (如 13-13c) 和 (2-13c)-甘油 (或 (1-13c)-和 (2-13c)-葡萄糖) 用于促进核磁共振分析。混合标记的样品对应于 50% 15N 和 50% 13c 标记或 50% (13-13c)-和 50% (2-13c)-葡萄糖的摩尔混合物, 以描述分子间的检测相互.在组装过程中, 高度的蛋白质纯度以及严格的条件是确保最终样品的均匀结构顺序的关键因素。
2. 基于 one-dimensional (1D) solid-state 核磁共振的初步结构表征
我们为 SSNMR 的结构分析提供了必要的实验。在13C 上检测到的一维 (1D) 极化 (CP) 和笨拙/RINEPT28实验, 分别用于检测组件中的刚性和挠性蛋白质片段, 并估计结构同质和局部多态性 (例如, 请参见图 3)。
<st></st>rong>3构象分析与3D 结构确定
第1和2节涉及的构象分析, 这是基于 SSNMR 共振分配的所有刚性残留的蛋白质组装, 因为化学转移是非常敏感的探针, 对当地环境和可用于预测的 phi/psi二面角, 从而决定第二结构。图 4说明了蛋白质组件的刚性内核中的顺序共振分配的示例.3D 结构的确定是基于结构数据的收集, 如距离限制编码关闭 proximities (和 #60; 7-9 Å), 包含两个内部和分子间的信息。第3和4节描述了远距离限制的收集和解释。远距离接触被定义为分子内的13c-13c proximities 产生于残留 i 到 j, 与 |4, 定义从而单体亚基的三重蛋白质折叠, 或者作为分子间的13c-13c proximities, 定义在组件中蛋白质亚基之间的分子之间的接口。在图 5中说明了内部和分子间的接口。SSNMR 的约束检测通过13c-13c 和15N13c 挂钩实验通常编码为核距离和 #60; 1 nm。4小节解释了分子间距离限制的检测。在对称的蛋白质组件中, 使用均匀标记的样本 (即100% 一致或有选择地标记) 来识别分子间亚基-亚基相互作用是有限的, 因为内部和分子接触导致探测信号。分子间 proximities 的明确检测是通过使用混合标记样品来实现的, 它包含了两种不同标记样品的摩尔混合物, 并在聚合之前结合在一起。5小节简要介绍了结构建模。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55779/55779fig1.jpg"> 
图 1:solid-state 核磁共振的原子解析结构研究的工作流.13C、 15N 同位素标记蛋白质生产、亚基纯化、亚基组装、装配形成控制、SSNMR 实验、SSNMR 实验分析和距离约束提取、结构建模等。<a href="//ecsource.jove.com/files/ftp_upload/55779/55779fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" width="741">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Instruments</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">NMR Spectrometer (&#62; 11.7 Tesla)</td>
			<td style="width:117px;">Bruker</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">triple resonance MAS SSNMR probehead&#160;</td>
			<td style="width:117px;">Bruker</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">SSNMR rotors 4mm</td>
			<td style="width:117px;">Bruker</td>
			<td style="width:119px;">K1910</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Centrifuge 5804 R&#160;</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:119px;">5805000629</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">GeneQuant 1300 spectrometer</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">28-9182-13</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">IGS60 INCUBATEUR HERATHERM 75 L</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">228001</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">MaxQ 4450 bench top orbital shaker</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">78376</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tube Revolver Agitator</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">79547</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">sonopuls HD 3100</td>
			<td style="width:117px;">Bandelin</td>
			<td style="width:119px;">3680</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">MicroPulser electroporator</td>
			<td style="width:117px;">Biorad</td>
			<td style="width:119px;">165-2100&#160;</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">mini-PROTEAN tetra cell system</td>
			<td style="width:117px;">Biorad</td>
			<td style="width:119px;">165-8000</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">AKTA pure system</td>
			<td style="width:117px;">GE Healthcare</td>
			<td style="width:119px;">29-0182-24</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">capillary microman M25 pipet</td>
			<td style="width:117px;">Gilson&#160;</td>
			<td style="width:119px;">F148502</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Materials</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:251px;">amiconR ultra-15</td>
			<td style="width:117px;">sigma</td>
			<td style="width:119px;">Z740199-8EA</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">capillaries and pistons</td>
			<td style="width:117px;">Gilson&#160;</td>
			<td style="width:119px;">F148112</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">spatula</td>
			<td style="width:117px;">Fisher</td>
			<td style="width:119px;">13263799</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Reagents</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:251px;">D-glucose 13C6</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">389374</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Ammonium-15N-chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">299251</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:251px;">1,3 13C2 glycerol</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">492639</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:251px;">2 13C glycerol</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">489484</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Kanamycin</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">K1876</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Carbenicillin</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">C3416</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sodium phosphate dibasic</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">S7907</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium phosphate monobasic</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">P5655</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sodium chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">71380</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">calcium chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">C1016</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Magnesium sulfate</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">208094</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iron Chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">157740</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Zinc chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">793523</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">MEM Vitamin Solution (100&#215;)</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">M68954</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">IPTG</td>
			<td style="width:117px;">Fisher</td>
			<td style="width:119px;">BP1755</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Trizma base&#160;</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricine</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">T0377</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">SDS</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">436143</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">sodium azide</td>
			<td style="width:117px;">sigma</td>
			<td style="width:119px;">71289</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">4,4-dimethyl-4-silapentane-1-sulfonic acid&#160;</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">178837</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Softwares</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Unicorn 6.3</td>
			<td style="width:117px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">Akta systems</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">ccpNMR</td>
			<td style="width:117px;">CCPN&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">spectrometer systems</td>
		</tr>
	</tbody>
</table>

atomic scale structural studies of macromolecular assemblies by solid-state nuclear magnetic resonance spectroscopy

超分子蛋白组件在生物过程中起着基本作用, 从宿主-病原体相互作用、病毒感染到神经退行性疾病的传播。此类组件由多个蛋白质亚基组成, 它们以价的方式组织, 形成大型大分子物, 可以执行多种细胞功能或造成有害后果。原子对组装机制和这些大分子组件的功能的洞察力仍然很稀少, 因为它们固有的不和 non-crystallinity 常常大大降低从大多数技术获得的数据的质量用于结构生物学, 如 x 射线晶体学和溶液核磁共振 (NMR)。我们在这里提出了魔术角旋转 solid-state 核磁共振光谱学 (SSNMR) 作为一个强有力的方法来研究大分子组件的结构在原子分辨率。SSNMR 可以在没有尺寸和溶解度限制的情况下揭示组装复合体的原子细节。此处介绍的协议描述了从生产13C/15N 同位素标记的大分子蛋白组件到获取标准 SSNMR 谱及其分析和解释的基本步骤。举例来说, 我们展示了一个丝状蛋白组装的 SSNMR 结构分析的管道。

典型的 SSNMR 工作流包括图 1中说明的几个步骤。通常, 蛋白质亚基是由体外产生的, 在大肠杆菌, 纯化和组装在震动, 但有时也在静态条件。蛋白质亚基的表达和纯化遵循 SDS 凝胶层析 (图 2A)。高分子组件的形成可以通过电子显微镜 (EM) 分析得到证实 (参见图 2B以得到一个丝状组件的例子)。
将该蛋白组件引入 SSNMR 转子后, 将转子插入光谱仪中, 对 MAS 频率和温度进行调节, 并记录频谱。第一个洞察力可以通过 1D SSNMR 技术获得。图 3显示了在结构均质蛋白质样本上的13c 通道上检测到的典型 SSNMR 裂, 一个1H-13c CP 频谱, 揭示了在蛋白质亚基的刚性核心中存在的13c 共振程序集和一个 2D 1H-13C 不称职的频谱, 表示移动残滓。为了原子洞察到装配结构的刚性核心, 多维 SSNMR 实验需要被记录在一致和有选择地标记的样品上, 首先分配 SSNMR 共振, 然后检测长程 proximities (参见图4).
所有的光谱都是用适当的软件来处理和分析的, 以分配 SSNMR 共振和提取内部和分子间距离限制 (图 5)。SSNMR 距离限制要么单独使用, 要么与互补技术的数据结合起来, 可以集成到建模程序中。
对于由 SSNMR 技术解决的大分子组件的代表性原子结构,图 6演示了从细菌附件和淀粉样纤维中得到的几个丝状组件。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55779/55779fig6v2.jpg"> 
图 6:由 solid-state 核磁共振方法确定的丝状大分子结构: 细菌丝和淀粉样蛋白纤维.A)类型1毛肾炎大肠杆菌, PDB 代码 2N7H 4;B) ASC 纤, PDB 代码 2N1F 63;C) III 型分泌系统针, PDB 代码 2MME, 2LPZ 和 2MEX 2,3,64;D) 淀粉样蛋白-β AB42 纤维, PDB 代码 2NAO, 5KK3, 2MXU 65,66,67和大阪变种 pdb 代码 2MVX 57, 爱荷华州变种 pdb 代码 2MPZ 58;E) α-核纤, PDB 代码 2N0A 68;F)-s 朊病毒域, PDB 代码 2RNM, 2KJ3 69,70。<a href="//ecsource.jove.com/files/ftp_upload/55779/55779fig6v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

超分子蛋白质的结构在原子分辨率下具有很高的相关性, 因为它们在各种生物现象中具有重要作用。在此, 我们提出了一个协议, 以执行高分辨率结构研究的不溶性和晶大分子蛋白组件的魔术角纺 solid-state 核磁共振光谱学 (MAS SSNMR)。

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

atomic-scale-structural-studies-macromolecular-assemblies-solid-state

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Analytical Chemistry

Biochemistry

Unit 1

Interpreting Nuclear Magnetic Resonance Spectra

SCIENTISTS IN ACTION

15.3K 次观看.  UMR5248 CNRS, Université de Bordeaux. 超分子蛋白质的结构在原子分辨率下具有很高的相关性, 因为它们在各种生物现象中具有重要作用。在此, 我们提出了一个协议, 以执行高分辨率结构研究的不溶性和晶大分子蛋白组件的魔术角纺 solid-state 核磁共振光谱学 (MAS SSNMR)。

视频: Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

核磁共振光谱法本质无序蛋白的磷酸化多元的鉴定

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

科研

生物化学

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

用核磁共振 (nmr) 和微尺度热能 (mst) 测量球状和丝状蛋白的相互作用

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

小角度 x 射线散射溶液中大分子的结构研究

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

基于核磁共振的片段筛选，样品最少，自动化程度最高

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...