Name: high-pressure nmr experiments for detecting protein low-lying conformational states
Uploaded: 2021-06-29T14:30:00
Description: Watch this Scientific Journal Video about High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States at JoVE.com

这项工作得到了罗伊·卡弗慈善信托基金和朱利安·罗氏基金的支持。我们感谢J.D.莱文古德和B.S.托尔伯特提供RRM2样本。

注：此处描述的协议要求 （i） 高压泵和电池具有 2.5 kbar 额定铝硬氧化锌管18，（ii）软件 SPARKY19 用于分析 NMR 光谱，（iii） 曲线拟合软件。
1. 样品制备、高压电池组装和设置实验。
<ol><li>缓冲器的选择：使用等离子和离子缓冲的混合物，如磷酸盐和三叶草20，21。 注：磷酸盐和 MES 等电离子...

<ol>
	<li>Alderson, R. T., 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=Unveiling+invisible+protein+states+with+NMR+spectroscopy.">Unveiling invisible protein states with NMR spectroscopy.</a> Current Opinion in Structural Biology. 60, 39-49 (2020).</li><li>Korzhnev, D. M., 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=probing+invisible,+low-populated+states+of+protein+molecules+by+relaxation+dispersion+NMR+spectroscopy:+An+application+to+protein+folding.">probing invisible, low-populated states of protein molecules by relaxation dispersion NMR spectroscopy: An application to protein folding.</a> Accounts of Chemical Research. 41, 442-451 (2008).</li><li>Loria, P. J., Berlow, R. B., Watt, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+enzyme+motions+by+solution+NMR+relaxation+dispersion.">Characterization of enzyme motions by solution NMR relaxation dispersion.</a> Accounts of Chemical Research. 41, 214-221 (2008).</li><li>Ishima, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPMG+relaxation+dispersion.">CPMG relaxation dispersion.</a> Methods in Molecular Biology. 1084, 29-49 (2014).</li><li>Longo, D. L., 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=Chemical+exchange+saturation+transfer+(CEST):+an+efficient+tool+for+detecting+molecular+information+on+proteins'+behaviour.">Chemical exchange saturation transfer (CEST): an efficient tool for detecting molecular information on proteins' behaviour.</a> Analyst. 39, 2687-2690 (2014).</li><li>Fawzi, N. L., Ying, J., Torchia, D. A., Clore, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+exchange+kinetics+and+atomic+resolution+dynamics+in+high-molecular-weight+complexes+using+dark-state+exchange+saturation+transfer+NMR+spectroscopy.">Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy.</a> Nature Protocols. 7, 1523-1533 (2012).</li><li>Anthis, N. J., Clore, M. G. <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, 35-116 (2015).</li><li>Roche, J., 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=Cavities+determine+the+pressure+unfolding+of+proteins.">Cavities determine the pressure unfolding of proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 6945-6950 (2012).</li><li>Chen, C. R., Makhatadze, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+determinant+of+the+effects+of+hydrostatic+pressure+on+protein+folding+stability.">Molecular determinant of the effects of hydrostatic pressure on protein folding stability.</a> Nature Communications. 8, 14561 (2017).</li><li>Roche, J., Royer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+pressure+denaturation+of+proteins.">Lessons from pressure denaturation of proteins.</a> Journal of the Royal Society Interface. 15, 20180244 (2018).</li><li>Xu, X., Gagn&#233;, D., Aramini, J. M., Gardner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+and+compressibility+differences+between+protein+conformations+revealed+by+high-pressure+NMR.">Volume and compressibility differences between protein conformations revealed by high-pressure NMR.</a> Biophysical Journal. 120, 924-935 (2021).</li><li>Akasaka, K., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-lying+excited+states+of+proteins+revealed+from+non-linear+pressure+shifts+in+1H+and+15N+NMR.">Low-lying excited states of proteins revealed from non-linear pressure shifts in 1H and 15N NMR.</a> Biochemistry. 40, 8665-8671 (2001).</li><li>Akasaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+conformational+fluctuation+of+proteins+by+pressure+perturbation.">Probing conformational fluctuation of proteins by pressure perturbation.</a> Chemical Reviews. 106, 1814-1835 (2006).</li><li>Kitahara, R., Yokoyama, S., Akasaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+snapshots+of+a+fluctuating+protein+structure:+ubiquitin+at+30+bar-3+kbar.">NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar-3 kbar.</a> Journal of Molecular Biology. 347, 277-285 (2005).</li><li>Roche, J., 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=remodeling+of+the+folding+free+energy+landscape+of+Staphylococcal+nuclease+by+cavity-creating+mutations.">remodeling of the folding free energy landscape of Staphylococcal nuclease by cavity-creating mutations.</a> Biochemistry. 51, 9535-9546 (2012).</li><li>Nucci, N. V., Fuglestad, B., Athanasoula, E. A., Wand, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cavities+and+hydration+in+the+pressure+unfolding+of+T4+lysozyme.">Role of cavities and hydration in the pressure unfolding of T4 lysozyme.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 13846-13851 (2014).</li><li>Maeno, A., 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=Cavity+as+a+source+of+conformational+fluctuation+and+high-energy+state:+High-pressure+NMR+study+of+a+cavity-enlarged+mutant+of+T4+lysozyme.">Cavity as a source of conformational fluctuation and high-energy state: High-pressure NMR study of a cavity-enlarged mutant of T4 lysozyme.</a> Biophysical Journal. 108, 133-145 (2015).</li><li>Peterson, R. W., Wand, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-contained+high-pressure+cell,+apparatus,+and+procedure+for+the+preparation+of+encapsulated+proteins+dissolved+in+low+viscosity+fluids+for+nuclear+magnetic+resonance+spectroscopy.">Self-contained high-pressure cell, apparatus, and procedure for the preparation of encapsulated proteins dissolved in low viscosity fluids for nuclear magnetic resonance spectroscopy.</a> Review of Scientific Instruments. 76, 094101 (2005).</li><li>Goddard, T. D., Kneller, D. 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> Sparky 3. , (2010).</li><li>Caro, J. A., Wand, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+high-pressure+NMR+spectroscopy+and+its+applications+in+protein+biophysics+and+structural+biology.">Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology.</a> Methods. 148, 67-80 (2018).</li><li>Kitamura, T., Itoh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+volume+of+protonic+ionization+for+buffering+agents.+Prediction+of+pressure+dependence+of+pH+and+pOH.">Reaction volume of protonic ionization for buffering agents. Prediction of pressure dependence of pH and pOH.</a> Journal of Solution Chemistry. 16, 715-725 (1987).</li><li>Royer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+volume+changes+in+pressure-induced+protein+unfolding.">Revisiting volume changes in pressure-induced protein unfolding.</a> Biochimica et Biophysica Acta. 1595, 201-209 (2002).</li><li>Erlach, M. B., 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=Relationship+between+nonliner+pressure-induced+chemical+shift+changes+and+thermodynamic+parameters.">Relationship between nonliner pressure-induced chemical shift changes and thermodynamic parameters.</a> Journal of Physical Chemistry B. 118, 5681-5690 (2014).</li><li>de Oliveira, G. A. P., Silva, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hypothesis+to+reconcile+the+physical+and+chemical+unfolding+of+proteins.">A hypothesis to reconcile the physical and chemical unfolding of proteins.</a> Proceedings of the National Academy of Sciences of The United States of America. 112, 2775-2784 (2015).</li><li>Nguyen, L. M., Roche, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+NMR+techniques+for+the+study+of+protein+dynamics,+folding+and+aggregation.">High-pressure NMR techniques for the study of protein dynamics, folding and aggregation.</a> Journal of Magnetic Resonance. 277, 179-185 (2017).</li></ol>

这项研究详细介绍了一项旨在探测蛋白质结构和热力学对压力扰动的反应的协议。RRM2上记录的高压实验表明，在相对较小的单一领域蛋白质中，可以发现+VU值的巨大变化，表明非完全合作展开。分析压力下的1H化学移位变化时，也出现了类似的情况。应当指出，卡尔比策和同事已经证明，可以对化学移位变化进行更深入的分析，将非线性和线性系数（B2/B1）与可压...

人们早就认识到，高能量、稀疏的蛋白质和蛋白质复合物的构象状态在许多生物途径1、2、3中起着关键作用。由于基于卡尔-珀塞尔-梅布姆-吉尔（CPMG）4、化学交换饱和转移（CEST）5和暗态交换饱和转移（DEST）6脉冲序列（等）的实验，解决方案NMR光谱学已成为描述瞬态构象状态7的一种选择方法。除了这些实验，可以引入温度、pH值或化学变性剂等扰动，以增加高能量构象亚态的相对数量。同样，蛋白质平衡也可以通过施加高静水压力来扰乱。根据与相应的构象变化相关的体积变化的大小，压力增加几百到几千条可以显著稳定更高的能量状态或导致蛋白质完全展开8，9，10。蛋白质NMR光谱通常显示两种类型的水静压变化：（i） 化学转移变化和（ii） 峰值强度变化。化学移位变化反映了蛋白质地表水界面的变化和/或蛋白质结构的局部压缩在快速的时间尺度（相对于NMR时间尺度）11。表现出大型非线性化学转移压力依赖性的十字说可以表明存在较高的能量构象状态12，13。另一方面，峰值强度变化指向缓慢时间尺度上的主要构象过渡，例如折叠/展开状态人口的变化。折叠中间体或更高能量状态的存在可以从体积变化幅度的巨大变化中检测到，当展开测量给定蛋白质的不同残留物14，15，16，17。根据我们的经验，即使是通常被归类为两州文件夹的微小蛋白质，对压力的反应也不均匀，这为当地折叠稳定性提供了有用的信息。这里描述的是获取和分析中层峰值强度和1H化学转移压力依赖性的协议，用作模型蛋白的分离RNA识别主题2（RRM2）的异质核核核蛋白A1（hnRNPA1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bruker Nmr Cell 2.5 Kbar</td>
			<td>Daedalus Innovations LLC</td>
			<td>NMRCELL-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sparky3</td>
			<td>University of California San Francisco, CA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Xtreme-60 Syringe pump</td>
			<td>Daedalus Innovations LLC</td>
			<td>XTREME-60</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-pressure nmr experiments for detecting protein low-lying conformational states

高压是一种众所周知的扰动方法，可用于破坏球状蛋白的稳定，并以可逆的方式分离蛋白质复合物。静水压力将热力学平衡推向摩尔体积较低的状态。因此，压力的增加提供了微调球蛋白稳定性和蛋白质复合物寡聚平衡的机会。高压NMR实验通过结合压力扰动的精细稳定性调谐能力和溶液NMR光谱提供的位点分辨率，对球蛋白稳定性、折叠机制和寡聚机制的因素进行了详细描述。在这里，我们提出了一个协议，通过一组从1巴记录到2.5千巴的2D 1H-15N实验来探索蛋白质的局部折叠稳定性。获取和分析此类实验所需的步骤用在 hnRNPA1 的 RRM2 域获得的数据来说明。

此处描述的协议用于调查 RRM2 的压力依赖性，RRM2 是 hnRNPA1 的第二个 RNA 识别主题（残留物 95-106），几乎完全展开于 2.5 kbar 范围内（>90%）。1H-15N光谱收集在1杆，500杆，750杆，1巴，1.5千巴，2公里，和2.5千巴（图2）。由于在2.5千巴的噪音水平以上看不到任何本地十字语，所有相应的残留物在这种压力下的强度值均为0（图3A）。根据方程 ...

Watch this Scientific Journal Video about High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States at JoVE.com

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

我们详细描述了组装高压电池所需的步骤，建立并记录了高压NMR实验，最后分析了压力下的峰值强度和化学移位变化。这些实验可以为蛋白质的折叠途径和结构稳定性提供有价值的见解。

检测蛋白质低洼构象状态的高压NMR实验

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible ...

高压是改变蛋白质构象样品相对量的选择方法。它对于隔离和描述高能构象状态非常有用。与所有扰动方法（如 pH 值或温度）相比，压力扰动易于应用且完全可逆。它也是局部扰动，主要影响玻璃腔或空量区域。首先，使用玻璃移液器将氮-15标签样品引入氧化锌管，并确保样品位于管底。为了防止样品与透水液混合，将样品与 200 微升矿物油覆盖，然后用透气液填充管子的其余部分。将一次性 O 环放在氧化锌管顶部，将管子滑入底座。然后将管子连接到高压系绳线，然后用手将底座拧紧到细胞上。然后应用 14.7 牛顿米扭矩，以防止在较低压力下泄漏。为了检查压力电池组件的完整性，使用电池支撑和密封容器对光谱仪外的管子加压至多 300 条。15 分钟后，将压力重置到一个栏，并使用干净的无绒毛擦拭检查泄漏情况。通过仔细引导系绳线，将未加压管插入光谱仪。将管子滑入光谱仪，直到管子达到样品坐姿。锁定，闪烁，匹配和调整的钚和氮通道，并保存优化的 shims 为未来。使用异质单量子相干光谱设置横向放松优化光谱，并在大气条件下进行记录参考实验。每 500 条记录一系列 2D 实验，从一个栏到 2.5 千巴。如果已知精确的折叠或展开速率，在每 500 条增量后，让样品均等化 15 到 20 分钟。当折叠或展开过渡的拐点到达时，记录额外的实验以提高拟合精度。该协议用于探讨RRM2的压力依赖性，RRM2是第二个RNA识别主题异质核核核蛋白A1。横向放松优化光谱与异质单量子相干光谱收集在一个酒吧，1.5千巴，两千巴和2.5千巴。RRM2 几乎完全展开在 2.5 千巴范围内。根据此处显示的方程安装单个压力强度配置文件，以获得与展开反应相关的标准 Gibbs 自由能量和体积的相应变化。在域结构上映射时，在域结构核心中发现了体积变化幅度最大的残留物，而体积变化幅度最小的残留物主要位于β链和β链与C终端氦的连接环中。为了探究折叠状态合奏的可压缩性和构象异质性，分析了蛋白化学移位。使用所示方程（提取特定地点的线性系数和非线性系数）来安装作为压力函数的蛋白化学移位的个别配置文件。非线性系数最大的残留物大多位于域的结构核心中，而非线性系数最小的残留物大多位于连接不同结构主题的环中。正确设置管子作为压力设置非常重要，以防止泄漏。如果发生泄漏，探测器可能需要拆除，以清理矿物油的任何痕迹。

Research

JoVE Journal

Biochemistry

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

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

用奇米蛋白构建功能蛋白区的鉴定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

优化用于连续晶体学实验的内硫肽晶体的生长

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

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

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 ...