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

人体可能含有超过200万种蛋白质，每个活细胞的每项功能都严重依赖于它们。蛋白质是由氨基酸组成的，这些氨基酸连接在一起，以各种组合（序列）排列成一条链。这个序列产生了一种独特的蛋白质，在体内具有不同的功能。在所有生命体中，细胞制造蛋白质的过程被称为翻译，是由高度复杂的微型工厂核糖体完成的——这些“机器”能够解码包含在我们遗传的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穿过被称为核糖体隧道的保护性通道，然后进入敌对的细胞环境。因此，这个NC试图保护自己，而不是像一个延伸的弦，它试图包裹或“折叠”成它的特征形状；这种形状赋予蛋白质不同的功能。指示NC折叠的信息包含在氨基酸序列中。然而，这个序列如何为折叠提供指导是生物学中的一个核心问题，它确实仍然是科学中最具争议的“圣杯”之一。蛋白质在细胞中快速有效地适应其形状是至关重要的。如果一个蛋白质链的序列编码错误，它可能会折叠成不正确的结构，后果可能是毁灭性的，导致II型糖尿病、阿尔茨海默氏症和帕金森症、疯牛病、囊性纤维化等疾病。了解蛋白质是如何折叠的，将使我们能够尝试逆转或防止它错误折叠的情况。目前，大多数研究都是在核糖体产生蛋白质后，在试管中检测蛋白质的折叠、三维结构和迁移性。虽然这让我们对蛋白质的行为有了一些不可思议的了解，但我们对这个过程在细胞内是如何发生的知之甚少。我们的兴趣集中在观察蛋白质链，因为它是在核糖体上形成的。我们建议在蛋白质链逐渐退出核糖体的过程中拍摄高分辨率的快照，以便我们了解它的发展。我们的部分建议是针对开发和实施新兴技术来生产这些快照-我们将使用基因工程工具对细菌细胞内的核糖体进行编程，使其在蛋白质制造过程中的不同阶段停止。接下来的策略是将核糖体从细胞中移除并放入试管中，之后我们可以使用一种称为核磁共振（NMR）的强大视觉技术来分析样本，这种技术可以在原子水平上检查蛋白质。其目的是为不同长度的蛋白质链拍摄许多快照，并展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所发生的一系列事件。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，我们将第一次以非常详细的方式描述蛋白质在细胞中形成时的结构，这是了解蛋白质在自然环境中折叠的重要一步，并有助于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来合理地设计小分子（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生。

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.