Structural investigation of co-translational folding events on the ribosome by NMR spectroscopy

通过核磁共振波谱研究核糖体上共翻译折叠事件的结构

• 批准号: BB/G015651/1
• 负责人: John Christodoulou
• 金额: $ 60.58万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG015651%2F1
• 关键词: Structural; investigation; co; translational; folding

The human body is likely to contain more than 2 million proteins, and every function in every living cell depends critically on them. Proteins are made up of building blocks called amino acids, which are linked together and arranged in various combinations (the sequence) to make a chain. The sequence results in a unique protein capable of a different function inside the body. In all kingdoms of life, protein manufacture by the cell occurs by a process known as translation and is carried out by highly sophisticated, miniature factories called ribosomes - these 'machines' are able to decode the instructions contained within our genetically inherited DNA blueprint and build a protein by adding amino acids one at a time to form the chain. During manufacture, the newly made chain, or 'nascent chain' (NC), journeys from the epicentre of these ribosome particles through a protective passage known as the ribosomal tunnel and then into the hostile cellular environment. This NC thus tries to protect itself so rather than resembling an extended string, it attempts to wrap up or 'fold' into its characteristic shape; the shape gives the protein a different function. The information which directs the NC folding is contained within the amino acid sequence. However, how this sequence provides instructions for folding is a central question in biology and it is indeed still one of the most hotly contested 'holy grails' of science. It is critical for a protein to adopt its shape quickly and efficiently in the cell. Should a protein chain have the wrong coding in its sequence it could fold into the incorrect structure and the consequences could be devastating, leading to diseases such as Type II Diabetes, Alzheimer's and Parkinson's, vCJD (Mad Cow disease), cystic fibrosis and many others. An understanding of how a protein folds, will allow us to try and reverse or prevent the occasions when it misfolds. At present, most studies have examined the folding and three-dimensional structure and mobility of a protein while it is in a test tube after its production by the ribosome. While this has given some incredible insights into protein behaviour, very little is known about how this process occurs within the cell. Our interest is centred on looking at the protein chain as it is being made on its ribosome. We propose to take high-resolution snapshots of the manufacture of a protein chain as it progressively exits the ribosome so that we can understand its development. A part of our proposal is geared towards developing and implementing emerging technologies to produce these snapshots - we will use the tools of genetic engineering to program the ribosomes within bacterial cells to halt at different stages during protein manufacture. This will be followed by a strategy to remove the ribosomes from the cells and into the test tube, after which we can analyze the sample using a powerful visual technique called Nuclear Magnetic Resonance (NMR), which can examine proteins at the level of the atom. The aim is to take many snapshots of protein chains of different lengths and showing a dynamic slideshow of the manufacturing process - the series of events that takes place from the time the protein chain leaves the ribosome until it forms its structure. We also plan to use these snapshots to understand what the ribosome looks like as it is making a new protein. This ambitious and challenging plan will mean that, for the first time and in extraordinary detail, we will be able to describe how a protein forms its structure when it is being made in the cell, a significant step closer to understanding protein folding in its natural environment and make inroads into why the misfolding processes described take place. This level of structural knowledge can also be used to rationally design small molecules (drugs) that can bind to a protein of interest and prevent any misfolding of the protein and as a consequence prevent the onset of disease states.

人体可能含有200多万种蛋白质，每个活细胞的每一项功能都取决于它们。蛋白质是由称为氨基酸的构建块组成的，氨基酸连接在一起并以各种组合（序列）排列形成链。该序列导致一种独特的蛋白质能够在体内发挥不同的功能。在所有的生命王国中，细胞制造蛋白质的过程被称为翻译，并由高度复杂的微型工厂进行，称为核糖体-这些“机器”能够解码包含在我们遗传遗传的DNA蓝图中的指令，并通过一次添加一个氨基酸形成链来构建蛋白质。在制造过程中，新制造的链或“新生链”（NC）从这些核糖体颗粒的中心通过称为核糖体隧道的保护通道，然后进入敌对的细胞环境。因此，这种NC试图保护自己，而不是像一个延长的字符串，它试图包裹或“折叠”成其特征形状;形状赋予蛋白质不同的功能。指导NC折叠的信息包含在氨基酸序列内。然而，该序列如何提供折叠指令是生物学中的一个核心问题，它确实仍然是科学界争议最激烈的“圣杯”之一。蛋白质在细胞中快速有效地改变其形状是至关重要的。如果蛋白质链在其序列中有错误的编码，它可能会折叠成不正确的结构，后果可能是毁灭性的，导致疾病，如II型糖尿病，阿尔茨海默氏症和帕金森氏症，vCJD（疯牛病），囊性纤维化和许多其他疾病。了解蛋白质如何折叠，将使我们能够尝试扭转或防止错误折叠的情况。目前，大多数研究都是在核糖体产生蛋白质后，在试管中研究蛋白质的折叠和三维结构以及运动性。虽然这为蛋白质行为提供了一些令人难以置信的见解，但人们对细胞内如何发生这一过程知之甚少。我们的兴趣集中在观察蛋白质链，因为它是在其核糖体上形成的。我们建议在蛋白质链逐渐离开核糖体时拍摄蛋白质链制造的高分辨率快照，以便我们能够了解其发展。我们的提案的一部分是针对开发和实施新兴技术来产生这些快照-我们将使用基因工程的工具来编程细菌细胞内的核糖体，使其在蛋白质制造的不同阶段停止。随后将采取一种策略，将核糖体从细胞中取出并放入试管中，之后我们可以使用称为核磁共振（NMR）的强大视觉技术分析样品，该技术可以在原子水平上检查蛋白质。其目的是拍摄不同长度的蛋白质链的许多快照，并显示制造过程的动态幻灯片-从蛋白质链离开核糖体到形成其结构的一系列事件。我们还计划利用这些快照来了解核糖体在制造新蛋白质时的样子。这一雄心勃勃且具有挑战性的计划将意味着，我们将能够第一次以非常详细的方式描述蛋白质在细胞中形成时如何形成其结构，这是理解蛋白质在其自然环境中折叠的重要一步，并深入了解所描述的错误折叠过程发生的原因。这种水平的结构知识也可以用于合理设计小分子（药物），这些小分子可以与感兴趣的蛋白质结合，防止蛋白质的任何错误折叠，从而防止疾病状态的发作。

项目成果

Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes.

• DOI: 10.1007/s10858-015-9968-x
• 发表时间: 2015-10
• 期刊: Journal of biomolecular NMR
• 影响因子: 2.7
• 作者: Chan SHS;Waudby CA;Cassaignau AME;Cabrita LD;Christodoulou J
• 通讯作者: Christodoulou J

1H, 15N and 13C assignments of domain 5 of Dictyostelium discoideum gelation factor (ABP-120) in its native and 8M urea-denatured states.

盘基网柄菌凝胶因子 (ABP-120) 的结构域 5 在其天然状态和 8M 尿素变性状态下的 1H、15N 和 13C 分配。

• DOI: 10.1007/s12104-008-9134-4
• 发表时间: 2009
• 期刊: Biomolecular NMR assignments
• 影响因子: 0.9
• 作者: Hsu ST

A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding.

• DOI: 10.1038/nsmb.3182
• 发表时间: 2016-04
• 期刊: Nature structural & molecular biology
• 影响因子: 16.8
• 作者: Cabrita LD;Cassaignau AME;Launay HMM;Waudby CA;Wlodarski T;Camilloni C;Karyadi ME;Robertson AL;Wang X;Wentink AS;Goodsell L;Woolhead CA;Vendruscolo M;Dobson CM;Christodoulou J
• 通讯作者: Christodoulou J

Transient Tertiary Structure Formation within the Ribosome Exit Port

• DOI: 10.1021/ja106530y
• 发表时间: 2010-12-01
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: O'Brien, Edward P.;Hsu, Shang-Te Danny;Dobson, Christopher M.
• 通讯作者: Dobson, Christopher M.