Name: 15n cpmg relaxation dispersion for the investigation of protein conformational dynamics on the &#181;s-ms timescale
Uploaded: 2021-04-19T15:00:00
Description: Watch this Scientific Journal Video about 15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the Ã‚Âµs-ms Timescale at JoVE.com

这项工作得到了来自NIGMS R35GM133488和罗伊·卡弗慈善信托基金到V.V.的资金支持。

1. 准备NMR样本
<ol><li>表达并净化2H，15N-实验室的感兴趣的蛋白质样本。 注：虽然 15个 N 标记蛋白质样本可用于获取 CPMG RD 实验，但渗透（在可能的情况下）可显著提高获得数据的质量。文献13提供了生产渗透蛋白的协议。</li>
	<li>缓冲区将纯化蛋白样品交换成脱气 NMR 缓冲器。</li>
	<li>将 NMR 样本传输到 NMR 管中。 注：NMR样本的浓度需要仔细优化，以...

<ol>
	<li>Anthis, N. J., Clore, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+transient+dark+states+by+NMR+spectroscopy.">Visualizing transient dark states by NMR spectroscopy.</a> Quarterly Reviews of Biophysics. 48 (1), 35-116 (2015).</li><li>Lisi, G. P., Loria, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+NMR+spectroscopy+for+the+study+of+enzyme+allostery.">Solution NMR spectroscopy for the study of enzyme allostery.</a> Chemical Reviews. 116 (11), 6323-6369 (2016).</li><li>Mittermaier, A., Kay, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+provide+new+insights+in+NMR+studies+of+protein+dynamics.">New tools provide new insights in NMR studies of protein dynamics.</a> Science. 312 (5771), 224-228 (2006).</li><li>Venditti, V., Clore, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+selection+and+substrate+binding+regulate+the+monomer/dimer+equilibrium+of+the+C-terminal+domain+of+Escherichia+coli+enzyme+I.">Conformational selection and substrate binding regulate the monomer/dimer equilibrium of the C-terminal domain of Escherichia coli enzyme I.</a> Journal of Biological Chemistry. 287 (32), 26989-26998 (2012).</li><li>Venditti, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+interdomain+rearrangement+triggered+by+suppression+of+micro-+to+millisecond+dynamics+in+bacterial+Enzyme+I.">Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I.</a> Nature Communications. 6, 5960 (2015).</li><li>Yip, G. N., Zuiderweg, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+cycle+scheme+that+significantly+suppresses+offset-dependent+artifacts+in+the+R2-CPMG+15N+relaxation+experiment.">A phase cycle scheme that significantly suppresses offset-dependent artifacts in the R2-CPMG 15N relaxation experiment.</a> Journal of Magnetic Resonance. 171 (1), 25-36 (2004).</li><li>Mulder, F. A., Skrynnikov, N. R., Hon, B., Dahlquist, F. W., Kay, L. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+TROSY+CPMG+sequence+for+characterizing+chemical+exchange+in+large+proteins.">A TROSY CPMG sequence for characterizing chemical exchange in large proteins.</a> Journal of Biomolecular NMR. 15 (2), 151-155 (1999).</li><li>Dotas, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+thermophilic/mesophilic+enzymes+reveal+a+role+for+conformational+disorder+in+regulation+of+bacterial+Enzyme+I.">Hybrid thermophilic/mesophilic enzymes reveal a role for conformational disorder in regulation of bacterial Enzyme I.</a> Journal of Molecular Biology. 432 (16), 4481-4498 (2020).</li><li>Purslow, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope+labeling+strategies+for+the+study+of+high-molecular-weight+proteins+by+solution+NMR+spectroscopy.">Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy.</a> Nature Protocols. 1 (2), 749-754 (2006).</li><li>Niklasson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+NMR+data+using+advanced+line+shape+fitting.">Comprehensive analysis of NMR data using advanced line shape fitting.</a> Journal of Biomolecular NMR. 69, 93-99 (2017).</li><li>Palmer, A. G., Kroenke, C. D., Loria, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+methods+for+quantifying+microsecond-to-millisecond+motions+in+biological+macromolecules.">Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules.</a> Methods in Enzymology. 339, 204-238 (2001).</li><li>Tollinger, M., Skrynnikov, N. R., Mulder, F. A., Forman-Kay, J. D., Kay, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+dynamics+in+folded+and+unfolded+states+of+an+SH3+domain.">Slow dynamics in folded and unfolded states of an SH3 domain.</a> Journal of the American Chemical Society. 123, 11341-11352 (2001).</li><li>Carver, J. P., Richards, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+two-site+solution+for+the+chemical+exchange+produced+dependence+of+T2+upon+the+Carr-Purcell+pulse+separation.">A general two-site solution for the chemical exchange produced dependence of T2 upon the Carr-Purcell pulse separation.</a> Journal of Magnetic Resonance. 6 (1), 89-105 (1972).</li><li>Egner, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Contrast'+NMR+Reveals+Non-innocent+Role+of+Support+in+Pd/CeO2+Catalyzed+Phenol+Hydrogenation.">Surface Contrast' NMR Reveals Non-innocent Role of Support in Pd/CeO2 Catalyzed Phenol Hydrogenation.</a> ChemCatChem. 12 (6), 4160-4166 (2020).</li><li>Egner, T. K., Naik, P., Nelson, N. C., Slowing, I. I., Venditti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+Insight+into+Nanoparticle+Surface+Adsorption+by+Solution+NMR+Spectroscopy+in+an+Aqueous+Gel.">Mechanistic Insight into Nanoparticle Surface Adsorption by Solution NMR Spectroscopy in an Aqueous Gel.</a> Angewandte Chemie (International Edition in English). 56, 9802-9806 (2017).</li><li>Tugarinov, V., Libich, D. S., Meyer, V., Roche, J., Clore, G. 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本手稿描述了在实验室中实施的协议，用于获取和分析15种 N RD 蛋白质数据。特别是，包括准备 NMR 样本、测量 NMR 数据和分析 RD 配置文件的关键步骤。下面讨论了有关研发实验的获取和分析的一些重要方面。然而，为了更深入地描述实验和数据分析，对原著文学的认真研究是强烈推荐的3、8、11、15、16。</s...

卡尔-珀塞尔·梅布姆-吉尔（CPMG）放松分散（RD）实验用于常规基础，通过溶液NMR光谱学1、2、3、4、5来描述在μs-ms时间尺度上发生的象形平衡。与其他构象动力学调查方法相比， CPMG 技术在现代 NMR 光谱仪上相对容易实现，不需要专门的样品制备步骤（即结晶、样品冻结或对齐、和/或与荧光或参数标记共价），并提供关于交换过程的构象平衡返回结构、动能和热力学信息的综合特征。为了让 CPMG 实验报告构象平衡，必须适用两个条件：（i） 观察到的 NMR 自旋必须在进行构象交换（微状态）的状态中具有不同的化学变化：（ii） 交换必须在从 ~50μs 到 ~10ms 的时间尺度上发生。在这些条件下，观察到的横向放松率 <img alt="Equation 1" src="/files/ftp_upload/62395/62395eq01v2.jpg"> （） 是内在 R2（在没有 μs-ms 动态的情况下测量的 R2） <img alt="Equation 2" src="/files/ftp_upload/62395/62395eq02v3.jpg"> 和对横向放松 （Rex）的交换贡献的总和。通过减少构成脉冲序列CPMG块的180°脉冲之间的间距，可以逐步消除对R2的研发贡献，并利用Bloch-McConnell理论对由此产生的RD曲线进行建模，以获得微态、每个微状态的微量人口和微状态之间的交换率（图1），2，3之间的化学移位差异。
在15项N CPMG实验的文献中，报告了几个不同的脉冲序列和分析方案。本文介绍了在实验室中实施的协议。特别是，将采用关键步骤，准备NMR样本，建立和获取NMR实验，以及处理和分析NMR数据（图2）。为了便于将协议传输到其他实验室，脉冲程序、处理和分析脚本以及一个示例数据集作为补充文件提供，可在 （https://group.chem.iastate.edu/Venditti/downloads.html） 下载。所提供的脉冲序列在 CPMG 块中包含一个四步相周期，用于抑制偏移依赖物件 6，并编码用于获取多个交错实验。这些交错的实验有一个相同的放松期，但不同数量的重新聚焦脉冲，以实现不同的CPMG领域7。同样重要的是要注意，所述脉冲程序测量了NMR信号8的TROSY组件的15N R2。 总体而言，该议定书已成功应用于大中型蛋白质4、5、9、10的构象交换定性。对于较小的系统（<20 kDa），建议使用基于异核单量子一致性（HSQC）的脉冲序列11，12。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cryoprobe</td>
			<td>Bruker</td>
			<td>5mm TCI 800 H-C/N-D cryoprobe</td>
			<td>Improve sensitivity</td>
		</tr>
		<tr>
			<td>Deuterium Oxide</td>
			<td>Sigma Aldrich</td>
			<td>756822-1</td>
			<td>&#62;99.8% pure, utilised in preparing NMR samples and deuterated cultures</td>
		</tr>
		<tr>
			<td>Hand driven centrifuge</td>
			<td>United Scientific supply</td>
			<td>CENTFG1</td>
			<td>Used to remove any air bubbles or residual liquid stuck on the walls of NMR tube.</td>
		</tr>
		<tr>
			<td>High Field NMR spectrometer</td>
			<td>Bruker</td>
			<td>Bruker Avance II 600, Bruker Avance 800</td>
			<td>acquisition of the NMR data</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>https://www.mathworks.com/products/get-matlab.html</td>
			<td>Modeling of the NMR data</td>
		</tr>
		<tr>
			<td>NMR pasteur Pipette</td>
			<td>Corning Incorporation</td>
			<td>7095D-NMR</td>
			<td>Pyrex glass pastuer pipette to transfer liquid sample in NMR tube</td>
		</tr>
		<tr>
			<td>NMR tube</td>
			<td>Willmad Precision</td>
			<td>535-PP-7</td>
			<td>5mm thin wall 7&#39;&#39; cylinderical glass tube</td>
		</tr>
		<tr>
			<td>NMRPipe</td>
			<td>Institute of Biosciences and Biotechnology research</td>
			<td>https://www.ibbr.umd.edu/nmrpipe/install.html</td>
			<td>NMR data processing</td>
		</tr>
		<tr>
			<td>SPARKY</td>
			<td>University of California, San Francisco</td>
			<td>https://www.cgl.ucsf.edu/home/sparky/</td>
			<td>Analysis of the NMR data</td>
		</tr>
		<tr>
			<td>Tospin 3.2 (or newer)</td>
			<td>Bruker</td>
			<td>https://www.bruker.com/protected/en/services/software-downloads/nmr/pc/pc-topspin.html</td>
			<td>acquisition software</td>
		</tr>
	</tbody>
</table>

15n cpmg relaxation dispersion for the investigation of protein conformational dynamics on the &#181;s-ms timescale

蛋白质构象动力学在调节酶催化、配体结合、异质和信号方面起着基础性作用，这些都是重要的生物过程。了解结构与动力学之间的平衡如何支配生物功能是现代结构生物学的一个新领域，并引发了多项技术和方法学的发展。其中，CPMG放松分散溶液NMR方法提供了关于在μs-ms时间尺度上发生的蛋白质相符合均衡的结构、动力学和热力学的独特原子分辨率信息。在这里，该研究提出了获取和分析15N放松分散实验的详细协议。例如，显示了用于分析细菌酶 I C 端域的 μs-ms 动力学的管道。

此处描述的协议导致在 1 H- 15N TROSY频谱（图 3A）中为每个峰值获取 RD 配置文件。从收购的RD配置文件，可以估计交换贡献的15N横向放松每个骨干阿米德组（图3A，3B）。通过绘制被调查蛋白质的3D结构的R前图，可以识别在μs-ms时间尺度（图3C）上进行构造交换的结构区域。使用卡弗-里?...

Watch this Scientific Journal Video about 15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the Ã‚Âµs-ms Timescale at JoVE.com

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the Ãƒâ€šÃ‚Âµs-ms Timescale

在这里，详细描述了在实验室中实施的协议，通过解决方案 NMR 光谱学获取和分析 15个 N 放松分散配置文件。

15N CPMG 放松分散，用于研究 μs-ms 时间尺度上的蛋白质符合性动力学

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

此协议可用于蛋白质构象动力学的检测和定性，这对理解各种细胞过程至关重要。与其他方法相比，该方法不需要专门的样品制备步骤，并提供了动力学、热力学和确认平衡结构方面的综合特征。CPMG放松分散可以更广泛地应用于核酸和其他生物分子中确认动力学的特征，以及配体纳米粒子相互作用的特征。我们的协议针对的是首次使用CPMG的用户。因此，这是一个很好的起点。但是，我们确实希望用户知道运行常规 NMR 实验的基本步骤。要首次设置 NMR 实验，请先下载并解压这些补充文件。复制位。vv和trosy_15N_CPMG。脉冲程序文件夹中的vv文件到脉冲程序目录。打开采集软件，使用 EDC 命令将以前运行的氢氮 15 HSQC 实验复制到新的实验中。使用脉冲程序命令加载trosy_15N_CPMG。vv脉冲程序文件进入新创建的实验。然后使用脉冲程序文件末尾的说明来设置 CPMG 实验。要设置常规 NMR 实验，将样品引入磁铁，并执行所有基本的 NMR 采集步骤。设置P1到氢硬90度脉冲的持续时间，P7设置为持续时间氮15硬90度脉冲。在采集窗口中，为氢气和氮气设置中心和光谱宽度 15 维。将放松延迟设置为 0.7 T2，并使用 VC 列表命令创建与 N 对应的整数列表。将 L8 设置为 VC 列表中的条目数量，将 L3 设置为间接维度的实际点数，将 1TD 到 L8 次 L3 设置为 2 倍。为了优化水抑制，将接收器增益设置为一个，并键入 EDC 拉开脉冲程序文件。在 91 行中，删除 goto 999 之前的半冒号，并保存文件。使用GS命令，调整SPDB0参数，以最大限度地降低FID信号的强度。修改信号强度后，在脉冲程序文件的第 91 行重新引入半冒号并保存文件。要优化 SPDB11，请将接收器增益设置为一个，并键入 EDC 拉动以打开脉冲程序文件。在 168 行中，删除 goto 999 之前的半冒号，并保存文件。使用GS命令，调整SPDB11参数，以最大限度地降低FID信号的强度。修改信号强度后，在 168 行重新引入半冒号并保存文件。要优化 SPDB2，将接收器增益设置为 1，然后输入 EDC 拉开脉冲程序文件。在 179 行上，删除 goto 999 之前的半冒号，并保存文件。使用GS命令，调整SPDB2参数，以最大限度地降低FID信号的强度。修改信号强度后，在脉冲程序文件的第 179 行重新引入半冒号并保存文件。要优化 PLDB2，请将接收器增益设置为 1，然后输入 EDC 拉开脉冲程序文件。在 184 行上，删除 goto 999 之前的半冒号，并保存文件。使用GS命令，调整PLDB2参数，以最大限度地降低FID信号的强度。修改信号强度后，在脉冲程序文件的第 184 行重新引入半冒号并保存文件。运行 RGA 命令以优化接收器增益。然后运行 ZG 命令以启动实验。在这个数字中，可以观察到氢氮15 Trosy光谱中每个峰值获得的放松分散剖面的结果。从获得的放松分散配置文件，可以估计交换贡献的氮15横向放松的每个骨干AMI组。通过绘制被调查的蛋白质3D结构的横向松弛图，可以识别在微秒毫秒时间尺度上进行确认交换的结构区域。使用卡弗-里查德方程对放松分散曲线进行建模，返回交换过程中的热力学和动能参数，例如处于均衡状态的状态的分数种群和这些状态之间的汇率。然后，可以分别使用面包车霍夫方程和I环方程来模拟这些热力学和动能参数的温度依赖性，以获得关于确认交换能量的详细信息。必须仔细优化所有收购参数，特别是P7。生产高度纯净和均质的样品也很重要，以避免虚假的分散。使用本协议获得的动能、热力学和化学芯片参数可用于获取有关进行确认交换的物种的能量和结构信息。CPMG方法获得的蛋白质确认动力学特征为理解信号和酶活性提供了重要信息，并为药物设计提供了新的视角。

Research

JoVE Journal

Biochemistry

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

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

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

Practical Aspects of Sample Preparation and Setup of 1H R1ÃƒÂÃ‚Â Relaxation Dispersion Experiments of RNA

1H R1 + RNA 放松分散实验样品制备和设置的实用方面

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...