Investigation into the structure dynamics and remodelling of the nucleosome using site-directed spin labelling and EPR distance measurement.

使用定点自旋标记和 EPR 距离测量研究核小体的结构动力学和重塑。

• 批准号: BB/E022286/1
• 负责人: David Norman
• 金额: $ 75.17万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE022286%2F1
• 关键词: Investigation; into; structure; dynamics; remodelling

The genomes of eukaryotes are packaged within the confines of the nucleus as a condensed structure termed chromatin. The nucleosome is the fundamental repeating subunit of chromatin. It consists of an octamer of four, core histone, proteins around which 146 bp of DNA is wrapped in nearly two turns. As a consequence all genetic processes in eukaryotes must contend with nucleosomes. For example, there are cellular mechanisms dedicated to modulating the dynamic properties of nucleosomes during the transcription cycle. These act to both improve and restrict access to the underlying genetic information as, and when, required. It is likely that the physical properties of the nucleosome are finely tuned to meet the apparently conflicting requirements of reducing inappropriate gene expression while allowing permitting transcription when required. Over the last ten years our understanding of the nucleosome has developed greatly due to the determination of X-ray crystal structures of the nucleosome. However, during assembly and remodelling, chromatin exists in different forms for which high resolution structures do not exist. Understanding of how chromatin structure is manipulated during the course of gene regulation is now limited by a lack of structural information regarding these intermediates. In order to address this a suitable technique would ideally be, carried out in aqueous solution (or frozen solution), be non-destructive (so we can add components in stages and look for change), provide accurate and suitable distances with limited interference to the underlying structure, and be able to give some indication of molecular dynamics. We propose to use a technique called Electron Paramagnetic Resonance (EPR) to study structure of a number of chromatin assemblies. Using established techniques we will introduce spin labels into specific places on either the histone proteins or DNA fragments. Spin labels provide signals in the EPR spectrum and by use of relatively new techniques we can measure the distance, between the spin labels, over distances of between approximately 2nm and 8nm. ( The diameter of the nucleosome is approximately 100A and its depth 50A) By triangulation these distance measurements allow us to build up a picture of the structure of the molecules containing the labels. In our experience of model systems, the distance measurements are likely to have an accuracy of around 0.1nm. This means that it will be possible to determine how changes in the composition of a complex like the nucleosome, lead to an overall changes in the structure of the complex. Such measurements will be made on the nucleosome and related structures in an attempt to fill vital details that are not described by the available crystal structures. Because the core of the nucleosome is made up of 4 protein dimers, the system is ideally suited to investigation by EPR but poses severe problems for a technique that could give similar measurements, called Fluorescence Resonance Energy Transfer (FRET). In EPR we can measure the distance between two identical spin labels. These labels are generally smaller, and less disruptive that the fluorescent labels used in FRET. As there are two copies of each histone protein in the nuclesome, it is technically very difficult to make nucleosomes labelled with single donor and acceptor dyes. In contrast, selection of a single labelling site on a dimeric histone that is separated by a suitable distance when assembled into a nucleosome, followed by spin labelling, provides a simple means of generating substrates labelled with two identical dyes that is suitable for EPR measurements. The most valuable information obtained by EPR is the measurement of distances between labelling sites. This is typically carried out in a frozen solution of between 10-200uM concentration. We have experience in using EPR to make distance measurements on DNA, Protein, DNA-protein and RNA-protein complexes.

真核生物的基因组被包装在细胞核的范围内，作为一个浓缩的结构称为染色质。核小体是染色质的基本重复亚基。它由四个八聚体组成，核心组蛋白，大约有146 bp的DNA以近两圈的方式包裹在蛋白质周围。因此，真核生物的所有遗传过程都必须与核小体竞争。例如，在转录周期中，有专门调节核小体动态特性的细胞机制。在需要的时候，这些措施既改善了对潜在遗传信息的获取，也限制了对潜在遗传信息的获取。核小体的物理性质很可能经过精细调整，以满足减少不适当基因表达的明显冲突要求，同时允许在需要时进行转录。在过去的十年中，由于对核小体的x射线晶体结构的测定，我们对核小体的认识有了很大的发展。然而，在组装和重塑过程中，染色质以不同的形式存在，而高分辨率结构不存在。由于缺乏关于这些中间体的结构信息，对基因调控过程中染色质结构如何被操纵的理解现在受到限制。为了解决这个问题，理想情况下，合适的技术应该是在水溶液（或冷冻溶液）中进行，非破坏性的（因此我们可以分阶段添加成分并寻找变化），提供准确和合适的距离，对底层结构的干扰有限，并且能够给出一些分子动力学的指示。我们建议使用一种称为电子顺磁共振（EPR）的技术来研究一些染色质组装的结构。利用现有的技术，我们将在组蛋白或DNA片段的特定位置引入自旋标签。自旋标签在EPR光谱中提供信号，通过使用相对较新的技术，我们可以测量自旋标签之间的距离，大约在2nm到8nm之间。（核小体的直径约为100A，深度约为50A）通过三角测量，这些距离测量使我们能够建立起包含标签的分子结构的图像。根据我们对模型系统的经验，距离测量的精度可能在0.1nm左右。这意味着将有可能确定像核小体这样的复合体的组成变化如何导致复合体结构的整体变化。这些测量将在核小体和相关结构上进行，试图填补现有晶体结构无法描述的重要细节。由于核小体的核心是由4个蛋白质二聚体组成的，该系统非常适合用EPR进行研究，但对一种可以进行类似测量的技术（称为荧光共振能量转移（FRET））提出了严重的问题。在EPR中，我们可以测量两个相同自旋标签之间的距离。这些标签通常比FRET中使用的荧光标签更小，破坏性更小。由于核小体中每个组蛋白都有两个拷贝，因此用单一的供体和受体染料标记核小体在技术上是非常困难的。相比之下，在二聚体组蛋白上选择一个标记位点，当组装成核小体时，该标记位点被分开适当的距离，然后进行自旋标记，提供了一种简单的方法，生成用两种相同的染料标记的底物，适用于EPR测量。EPR获得的最有价值的信息是测量标记点之间的距离。这通常在10-200uM浓度的冷冻溶液中进行。我们有使用EPR对DNA、蛋白质、DNA-蛋白质和rna -蛋白质复合物进行距离测量的经验。

项目成果

Quantitative analysis of chromatin compaction in living cells using FLIM-FRET.

• DOI: 10.1083/jcb.200907029
• 发表时间: 2009-11-16
• 期刊: The Journal of cell biology
• 作者: Llères D;James J;Swift S;Norman DG;Lamond AI
• 通讯作者: Lamond AI

The spatial effect of protein deuteration on nitroxide spin-label relaxation: implications for EPR distance measurement.

• DOI: 10.1016/j.jmr.2014.09.010
• 发表时间: 2014-11
• 期刊: JOURNAL OF MAGNETIC RESONANCE
• 影响因子: 2.2
• 作者: El Mkami, Hassane;Ward, Richard;Bowman, Andrew;Owen-Hughes, Tom;Norman, David G.
• 通讯作者: Norman, David G.

DNA repair factor APLF is a histone chaperone.

• DOI: 10.1016/j.molcel.2010.12.008
• 发表时间: 2011-01-07
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Mehrotra PV;Ahel D;Ryan DP;Weston R;Wiechens N;Kraehenbuehl R;Owen-Hughes T;Ahel I
• 通讯作者: Ahel I

The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3-H4 tetramers and Asf1-H3-H4 complexes.

• DOI: 10.1093/nar/gkw209
• 发表时间: 2016-07-27
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Hammond CM;Sundaramoorthy R;Larance M;Lamond A;Stevens MA;El-Mkami H;Norman DG;Owen-Hughes T
• 通讯作者: Owen-Hughes T

Probing the (H3-H4)2 histone tetramer structure using pulsed EPR spectroscopy combined with site-directed spin labelling.

使用脉冲EPR光谱与位置定向的自旋标记探测（H3-H4）2组蛋白四聚体结构。

• DOI: 10.1093/nar/gkp1003
• 发表时间: 2010-01
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Bowman A;Ward R;El-Mkami H;Owen-Hughes T;Norman DG
• 通讯作者: Norman DG