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><tbody><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NMR Spectrometer (&gt; 11.7 Tesla)</td><td>Bruker</td><td>-</td><td></td></tr><tr><td>triple resonance MAS SSNMR probehead&amp;nbsp;</td><td>Bruker</td><td>-</td><td></td></tr><tr><td>SSNMR rotors 4mm</td><td>Bruker</td><td>K1910</td><td></td></tr><tr><td>Centrifuge 5804 R&amp;nbsp;</td><td>Eppendorf</td><td>5805000629</td><td></td></tr><tr><td>GeneQuant 1300 spectrometer</td><td>Dutscher</td><td>28-9182-13</td><td></td></tr><tr><td>IGS60 INCUBATEUR HERATHERM 75 L</td><td>Dutscher</td><td>228001</td><td></td></tr><tr><td>MaxQ 4450 bench top orbital shaker</td><td>Dutscher</td><td>78376</td><td></td></tr><tr><td>Tube Revolver Agitator</td><td>Dutscher</td><td>79547</td><td></td></tr><tr><td>sonopuls HD 3100</td><td>Bandelin</td><td>3680</td><td></td></tr><tr><td>MicroPulser electroporator</td><td>Biorad</td><td>165-2100&amp;nbsp;</td><td></td></tr><tr><td>mini-PROTEAN tetra cell system</td><td>Biorad</td><td>165-8000</td><td></td></tr><tr><td>AKTA pure system</td><td>GE Healthcare</td><td>29-0182-24</td><td></td></tr><tr><td>capillary microman M25 pipet</td><td>Gilson&amp;nbsp;</td><td>F148502</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>amicon&lt;sup&gt;R&lt;/sup&gt; ultra-15</td><td>sigma</td><td>Z740199-8EA</td><td></td></tr><tr><td>capillaries and pistons</td><td>Gilson&amp;nbsp;</td><td>F148112</td><td></td></tr><tr><td>spatula</td><td>Fisher</td><td>13263799</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D-glucose &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>389374</td><td></td></tr><tr><td>Ammonium-15N-chloride</td><td>Sigma</td><td>299251</td><td></td></tr><tr><td>1,3 &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;2&lt;/sub&gt; glycerol</td><td>Sigma</td><td>492639</td><td></td></tr><tr><td>2 &lt;sup&gt;13&lt;/sup&gt;C glycerol</td><td>Sigma</td><td>489484</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma</td><td>K1876</td><td></td></tr><tr><td>Carbenicillin</td><td>Sigma</td><td>C3416</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>calcium chloride</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>208094</td><td></td></tr><tr><td>Iron Chloride</td><td>Sigma</td><td>157740</td><td></td></tr><tr><td>Zinc chloride</td><td>Sigma</td><td>793523</td><td></td></tr><tr><td>MEM Vitamin Solution (100&amp;times;)</td><td>Sigma</td><td>M68954</td><td></td></tr><tr><td>IPTG</td><td>Fisher</td><td>BP1755</td><td></td></tr><tr><td>Trizma base&amp;nbsp;</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>436143</td><td></td></tr><tr><td>sodium azide</td><td>sigma</td><td>71289</td><td></td></tr><tr><td>4,4-dimethyl-4-silapentane-1-sulfonic acid&amp;nbsp;</td><td>Sigma</td><td>178837</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Unicorn 6.3</td><td>GE Healthcare</td><td></td><td>Akta systems</td></tr><tr><td>ccpNMR</td><td>CCPN&amp;nbsp;</td><td></td><td>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

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Analytical Chemistry

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Unit 1

Interpreting Nuclear Magnetic Resonance Spectra

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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

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 ...

科研

生物化学

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 ...

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely M&#246;ssbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. M&#246;ssbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

Here, we present a protocol to describe ex situ and in situ investigations of structural transformations in metallic glasses. We employed nuclear-based analytical methods which inspect hyperfine interactions. We demonstrate the applicability of M&#246;ssbauer spectrometry and nuclear forward scattering of synchrotron radiation during temperature-driven experiments.

结构转换的原位和原位研究方法: 金属玻璃结晶的实例

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic ...

工程学

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There exists a drive to characterize materials under conditions relevant to the chemical process of interest. To address this, in situ high-temperature, high-pressure MAS NMR methods have been developed to enable the observation of chemical interactions over a range of pressures (vacuum to several hundred bar) and temperatures (well below 0 &#176;C to 250 &#176;C). Further, the chemical identity of the samples can be comprised of solids, liquids, and gases or mixtures of the three. The method incorporates all-zirconia NMR rotors (sample holder for MAS NMR) which can be sealed using a threaded cap to compress an O-ring. This rotor exhibits great chemical resistance, temperature compatibility, low NMR background, and can withstand high pressures. These combined factors enable it to be utilized in a wide range of system combinations, which in turn permit its use in diverse fields as carbon sequestration, catalysis, material science, geochemistry, and biology. The flexibility of this technique makes it an attractive option for scientists from numerous disciplines.

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

高温高压原位魔角旋转核磁共振光谱

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There ...

化学

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

<meta charset="utf8"></head><body>蛋白质动力学研究中 NMR 15N 弛豫和 hetNOE 实验的方案

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

<meta charset="utf8"></head><body>作者聚焦：通过 NMR 弛豫实验探索内在无序的蛋白质动力学

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

NMR 15N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

固体核磁共振波谱法研究高分子组件的原子尺度结构

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此视频中的章节

小角度 x 射线散射溶液中大分子的结构研究

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

结构转换的原位和原位研究方法: 金属玻璃结晶的实例

高温高压原位魔角旋转核磁共振光谱

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

NMR ¹⁵N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

NMR ¹⁵N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学

NMR ¹⁵N 弛豫实验，用于研究蛋白质皮秒至纳秒的结构动力学