体外 使用分析、下拉和伴侣测定表征组蛋白伴侣
Summary
该协议描述了一系列方法，包括分析体积排阻色谱以研究组蛋白伴侣寡聚化和稳定性，下拉测定以揭示组蛋白伴侣 – 组蛋白相互作用，AUC用于分析蛋白质复合物的化学计量，以及组蛋白伴侣测定以在 体外功能表征假定的组蛋白伴侣。
Abstract
组蛋白与DNA结合形成真核染色质。染色质的基本单位是核小体，由组蛋白八聚体组成，由核心组蛋白H2A，H2B，H3和H4的两个拷贝组成，被DNA包裹。八聚体由两个拷贝的H2A/H2B二聚体和一个拷贝的H3/H4四聚体组成。高电荷的核心组蛋白容易与细胞质和细胞核中的几种蛋白质发生非特异性相互作用。组蛋白伴侣形成一类多样化的蛋白质，将组蛋白从细胞质穿梭到细胞核中，并帮助它们沉积到DNA上，从而协助核小体组装过程。一些组蛋白伴侣对 H2A/H2B 或 H3/H4 具有特异性，有些则作为两者的伴侣。该协议描述了如何串联使用体 外 实验室技术，例如下拉测定，分析体积排阻色谱，分析超离心和组蛋白伴侣测定，以确认给定的蛋白质是否具有组蛋白伴侣的功能。
Introduction
由DNA和组蛋白组成的核小体形成染色质的结构单元并调节几个关键的细胞事件。核小体被动态地重新定位和重塑，使DNA能够被复制、转录和翻译等各种过程所访问1，2。高度碱性的组蛋白要么倾向于与细胞环境中的酸性蛋白质相互作用，要么发生聚集，从而导致各种细胞缺陷3，4，5。一组称为组蛋白伴侣的专用蛋白质有助于组蛋白从细胞质转运到细胞核，并防止异常组蛋白-DNA聚集事件6，7。从根本上说，大多数组蛋白伴侣以生理离子强度将组蛋白储存并转移到DNA上，从而有助于核小体8，9的形成。一些组蛋白伴侣对组蛋白低聚物H2A/H2B或H3/H410有明确的偏好。
组蛋白伴侣的特征基于它们组装依赖或独立于DNA合成的核小体的能力11。例如，染色质组装因子-1 （CAF-1） 是依赖性的，而组蛋白调节因子 A （HIRA） 独立于 DNA 合成12，13。同样，组蛋白伴侣的核质蛋白家族参与精子染色质脱缩和核小体组装14。核小体组装蛋白（NAP）家族成员在体外促进核小 体 样结构的形成，并参与细胞质和细胞核之间的组蛋白穿梭15。核质蛋白和NAP家族蛋白都是功能性组蛋白伴侣，但没有任何结构特征。从本质上讲，没有单一的结构特征允许将蛋白质分类为组蛋白伴侣16。使用功能和生物物理测定以及结构研究最适合表征组蛋白伴侣。
这项工作描述了将蛋白质表征为帮助核小体组装的组蛋白伴侣的生物化学和生物物理方法。首先，采用分析体积排阻色谱法分析组蛋白伴侣的寡聚状态和稳定性。接下来，进行下拉测定以确定组蛋白伴侣-组蛋白相互作用的驱动力和竞争性质。然而，由于蛋白质的形状及其复合物会影响它们在色谱柱中的迁移，因此使用分析体积排阻色谱无法准确计算这些相互作用的流体动力学参数。因此，使用分析超速离心，它提供了溶液内大分子特性，包括准确的分子量、相互作用的化学计量和生物分子的形状。过去的研究已广泛使用体外组蛋白伴侣测定来功能表征组蛋白伴侣，例如yScS116 17，DmACF18，ScRTT106p19，HsNPM120。组蛋白伴侣测定也用于在功能上表征蛋白质作为组蛋白伴侣。
Protocol
1. 分析体积排阻色谱法阐明组蛋白伴侣的低聚状态和稳定性 组蛋白伴侣寡聚状态分析在4 °C下平衡24 mL分析体积排阻色谱（SEC）柱与1.2柱体积（CV），即28.8 mL脱气SEC缓冲液[20 mM的Tris-HCl（pH 7.5），300 mM NaCl和1 mM的β-巯基乙醇（β-ME）]（参见 材料表）。注意：色谱柱类型、缓冲液组成和缓冲液pH值可根据感兴趣的蛋白质进行选择。对于24 mL色谱柱，样品进样?…
Representative Results
对拟 南芥 FKBP53蛋白的重组N端核质结构域进行SEC分析。将洗脱峰体积与标准曲线作图，以确定其低聚状态。SEC分析结果表明，该结构域以五聚体的形式存在于溶液中，分子量约为58 kDa（图1A，B）。此外，结合分析SEC分析核蓝蛋白结构域的热稳定性和化学稳定性。与保持在20 °C的样品相比，在高达90 °C的温度下进行热处理的核蓝蛋白样品的洗脱体积和峰高?…
Discussion
这项工作演示并验证了一套全面的方案，用于推定组蛋白伴侣的生化和生物物理表征。本文使用重组表达和纯化的NAP家族蛋白AtNRP1和AtNRP2以及AtFKBP53的N端核质蛋白结构域来证明方案。同一组实验可以很好地用于描述来自任何生物体的以前未表征的组蛋白伴侣的功能属性。
协议部分的第一部分涉及研究组蛋白伴侣的寡聚状态和稳定性。一些报告表明，组蛋白伴侣在其寡聚状态?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
印度政府科学与工程研究委员会[CRG/2018/000695/PS]和印度政府科学技术部生物技术司[BT/INF/22/SP33046/2019]向Dileep Vasudevan提供的校外赠款，以及布巴内斯瓦尔生命科学研究所的校内支持，都得到了极大的认可。我们感谢Sudeshna Sen女士和Annapurna Sahoo女士在组蛋白制备方面的帮助。与我们的同事钦马伊·莫哈帕特拉博士、玛纳斯·库马尔·贾格德夫先生和谢赫·瑙萨德·侯赛因博士的讨论也得到了认可。
Materials
| Acetic acid (glacial) | Sigma | A6283 | |
| Acrylamide | MP Biomedicals | 814326 | |
| Agarose | MP Biomedicals | 193983 | |
| AKTA Pure 25M FPLC | Cytiva | 29018226 | Instrument for protein purification |
| Ammonium persulfate (APS) | Sigma | A3678 | |
| An-60Ti rotor | Beckman Coulter | 361964 | Rotor for analytical ultracentrifugation |
| Bovine serum albumin (BSA) | Sigma | A7030 | |
| Chloroform | Sigma | C2432 | |
| Coomassie brilliant blue R 250 | Sigma | 1.15444 | |
| Dialysis tubing (7 kDa cut-off) | Thermo Fisher | 68700 | For dialysing protein samples |
| Dithiothreitol (DTT) | MP Biomedicals | 100597 | |
| DNA Loading Dye | New England Biolabs | B7025S | |
| EDTA disodium salt | MP Biomedicals | 194822 | |
| Electronic balance | Shimadzu | ATX224R | |
| Ethanol | Sigma | E7023 | |
| Ethidium bromide (EtBr) | Sigma | E8751 | |
| Gel Doc System | Bio-Rad | 12009077 | For imaging gels after staining |
| Horizontal gel electrophoresis apparatus | Bio-Rad | 1704405 | Instrument for agarose gel electrophoresis |
| Hydrochloric acid (HCl) | Sigma | 320331 | |
| Imidazole | MP Biomedicals | 102033 | |
| Magnesium chloride (MgCl2) | Sigma | M8266 | |
| Micropipettes | Eppendorf | Z683779 | For pipetting of micro-volumes |
| Mini-PROTEAN electrophoresis system | Bio-Rad | 1658000 | Instrument for SDS-PAGE |
| N,N-methylene-bis-acrylamide | MP Biomedicals | 800172 | |
| Nano drop | Thermo Fisher | ND-2000 | For measurement of protein and DNA concentrations |
| Ni-NTA agarose | Invitrogen | R901-15 | Resin beads for pull-down assay |
| Optima AUC analytical ultracentrifuge | Beckman Coulter | B86437 | Instrument for analytical ultracentrifugation |
| pH Meter | Mettler Toledo | MT30130863 | |
| Phenol | Sigma | P4557 | |
| Plasmid isolation kit | Qiagen | 27104 | |
| Proteinase K | Sigma-Aldrich | 1.07393 | |
| pUC19 | Thermo Fisher | SD0061 | Plasmid for supercoiling assay |
| Refrigerated high-speed centrifuge | Thermo Fisher | 75002402 | |
| SDS-PAGE protein marker | Bio-Rad | 1610317 | |
| SEDFIT | Free software program for analytical ultracentrifugation data analysis | ||
| SEDNTERP | Free software program to estimate viscosity and density of buffer and partial specific volume of a protein | ||
| SigmaPrep Spin Columns | Sigma | SC1000 | For pull-down assay |
| Sodium acetate | Sigma | S2889 | |
| Sodium chloride (NaCl) | Merck | S9888 | |
| Sodium dodecyl sulfate (SDS) | MP Biomedicals | 102918 | |
| Superdex 200 Increase 10/300 GL | Cytiva | 28990944 | Column for analytical size-exclusion chromatography |
| Superdex 75 Increase 10/300 GL | Cytiva | 29148721 | Column for analytical size-exclusion chromatography |
| TEMED | Sigma | 1.10732 | |
| Topoisomerase I | Inspiralis | WGT102 | Enzyme used in plasmid supercoiling assay |
| Tris base | Merck | T1503 | |
| Tween-20 | Sigma | P1379 | |
| Urea | MP Biomedicals | 191450 | |
| Water bath | Nüve | NB 5 | For heat treatment of protein samples |
| β-mercaptoethanol (β-ME) | Sigma | M6250 |
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Cite This Article
Bobde, R. C., Saharan, K., Baral, S., Gandhi, S., Samal, A., Sundaram, R., Kumar, A., Singh, A. K., Datta, A., Vasudevan, D. In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays. J. Vis. Exp. (178), e63218, doi:10.3791/63218 (2021).