The molecular mechanisms determining the onset of protein aggregation revealed by single molecule force-clamp spectroscopy

单分子力钳光谱揭示决定蛋白质聚集开始的分子机制

• 批准号: BB/J00992X/1
• 负责人: Sergi Garcia-Manyes
• 金额: $ 46.73万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ00992X%2F1
• 关键词: molecular; mechanisms; determining; onset; protein

Each organ in our body is composed of a large number of individual cells working together in a coordinated fashion. Inside each cell, there are thousands of different proteins that perform their function in a very well-established and synchronized way. In general, each of these proteins can be found in two different shapes -the folded and the unfolded states. Proteins unfold and refold continuously in our bodies once they are expressed in the ribosomes, which are the small factories where they are produced. Most proteins are 'active' or 'functional' only when they are in their folded state. Failing to fold gives rise to a myriad of devastating diseases such as Alzhemier's, Parkinson's, Mad Cow, eye's cataracts and many others. These diseases have their origin when a single molecule undergoes a conformational change and is not able to fold back into its native structure anymore. This is one of the origins of toxicity; once a protein is unfolded and cannot get back to its folded state, it sticks to a neighboring unfolded protein in a rather fast way. This creates a nucleation seed that eventually results in the presence of aggregates, called amyloids, which are the signature of several diseases. For example, plaques of aggregates are found in the brains of Alzhemier's and Parkinson's disease patients. Perhaps the most conspicuous amyloid disease is cataract formation in the human eye, where the amyloids can be observed directly by looking into an affected eye. Unfortunately, once these amyloid aggregates are detected, it is often too late to act. It is therefore really challenging to discover why and when this aggregative process starts. This project aims to develop a new strategy to able to experimentally manipulate the state of a single protein and probe the time evolution of the different shapes and conformations adopted by each protein during its folding trajectory. The challenge is now to detect the mechanisms why proteins fail to fold, at the level of a single molecule. There are very few and only recent studies of aggregating proteins at the single molecule level. I will use a novel technique, named single molecule force-clamp spectroscopy, to study the different trajectories followed by an unfolded protein in its journey to the native state. This technique has already proved successful at identifying, for the first time, the different conformations adopted by a protein that has been evolutionary designed to fold. I will first further the investigations to completely understand the different trajectories followed by an individual protein until it folds. I will then expand this methodology to study the folding behaviour of proteins that cause a great variety of diseases such as Alzheimer's (the Abeta42 polypeptide), Parkinson's (caused by the aggregation of the alpha-synuclein protein) and also the eye's cataract, which is triggered by the misfolding of the protein gammaD crystallin. In all these cases, I will compare the behaviour of these amyloid-forming proteins with those proteins that succeed to fold. I will identify the conformation (folded, unfolded or intermediate) where each of these amyloid-forming proteins departs from the 'functional' folding route. I will finally study if the presence of other companion proteins, called chaperones, assists in the folding mechanism of the aggregating proteins. Altogether, these single molecule techniques have now reached a level of maturity where they can be used to attack more significant challenges in biology such as the basic biological mechanisms leading to protein aggregation, originating at the single molecule level. I seize on the remarkable opportunity of expanding the current applications of force-clamp to solving the common riddle of these diseases. These basic biophysical studies hold great promise for impacting several fields of research, such as the molecular understanding of such devastating human diseases for which there is, at the present time, no cure.

我们体内的每个器官都由大量以协调方式一起工作的单个单元组成。在每个细胞中，有成千上万种不同的蛋白质以非常有成就和同步的方式执行其功能。通常，这些蛋白质中的每一个都可以以两种不同的形状（折叠状态和展开状态）找到。蛋白质一旦在核糖体中表达，这是它们产生的小工厂，就会在我们的体内持续重新散发。大多数蛋白质仅在其折叠状态时才是“活跃”或“功能”。未能折叠会导致无数毁灭性的疾病，例如阿尔茨米尔，帕金森氏症，疯牛，眼睛的白内障等。当单个分子经历构象变化并且无法再折叠到其天然结构时，这些疾病具有起源。这是毒性的起源之一；一旦展开蛋白质并且无法回到其折叠状态，它就会以相当快的方式粘在相邻的展开蛋白质上。这产生了成核种子，最终导致聚集体的存在，称为淀粉样蛋白，这是几种疾病的特征。例如，在阿尔茨米尔和帕金森氏病患者的大脑中发现了骨料的斑块。也许最明显的淀粉样蛋白疾病是人眼中的白内障形成，可以直接观察到受影响的眼睛，可以直接观察淀粉样蛋白。不幸的是，一旦检测到这些淀粉样蛋白聚集体，采取行动通常为时已晚。因此，发现为什么以及何时开始这种聚合过程是真正的挑战。该项目旨在制定一种新策略，以实验操纵单蛋白的状态并探测每个蛋白在其折叠轨迹期间采用的不同形状和构象的时间演变。现在的挑战是检测蛋白质在单个分子水平上无法折叠的机制。在单分子水平上汇总蛋白质的研究很少，而且只有最近的研究。我将使用一种名为单分子力钳光谱的新型技术来研究不同的轨迹，然后在其前往本地状态的过程中进行了展开的蛋白质。该技术已经证明成功地识别了一种旨在折叠的蛋白质所采用的不同构象。我将首先进行研究，以完全了解不同的轨迹，然后是单个蛋白质，直到折叠为止。然后，我将扩展这种方法论，以研究蛋白质的折叠行为，这些蛋白质引起了多种疾病，例如阿尔茨海默氏症（Abeta42多肽），帕金森氏症（由α-阴性核蛋白蛋白的聚集引起的）以及眼神cata虫的cylet虫，这些蛋白质是由蛋白质粘附的脱落而触发的。在所有这些情况下，我将将这些淀粉样蛋白的行为与成功折叠的蛋白质进行比较。我将确定构象（折叠，展开或中间体），其中这些淀粉样蛋白中的每一个都从“功能”折叠路线偏离。我最终将研究其他伴随蛋白（称为伴侣）的存在有助于聚集蛋白的折叠机理。总的来说，这些单分子技术现在已经达到了成熟水平，可以用来攻击生物学中更大的挑战，例如导致蛋白质聚集的基本生物学机制，起源于单分子水平。我抓住了扩大力钳在解决这些疾病的共同谜语中的当前应用的非凡机会。这些基本的生物物理研究对影响几个研究领域的巨大希望，例如对目前无法治愈的这种毁灭性人类疾病的分子理解。

项目成果

Steering chemical reactions with force

• DOI: 10.1038/s41570-017-0083
• 发表时间: 2017-11-01
• 期刊: NATURE REVIEWS CHEMISTRY
• 影响因子: 36.3
• 作者: Garcia-Manyes, Sergi;Beedle, Amy E. M.
• 通讯作者: Beedle, Amy E. M.

The force-dependent mechanism of DnaK-mediated mechanical folding.

• DOI: 10.1126/sciadv.aaq0243
• 发表时间: 2018-03
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Perales-Calvo J;Giganti D;Stirnemann G;Garcia-Manyes S
• 通讯作者: Garcia-Manyes S

Dividing cells regulate their lipid composition and localization.

• DOI: 10.1016/j.cell.2013.12.015
• 发表时间: 2014-01-30
• 期刊: Cell
• 影响因子: 64.5
• 作者: Atilla-Gokcumen GE;Muro E;Relat-Goberna J;Sasse S;Bedigian A;Coughlin ML;Garcia-Manyes S;Eggert US
• 通讯作者: Eggert US

The Mechanochemistry of a Structural Zinc Finger.

• DOI: 10.1021/acs.jpclett.5b01371
• 发表时间: 2015-08
• 期刊: The journal of physical chemistry letters
• 作者: Judit Perales-Calvo;Ainhoa Lezamiz;S. Garcia-Manyes

Tailoring protein nanomechanics with chemical reactivity.

• DOI: 10.1038/ncomms15658
• 发表时间: 2017-06-06
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Beedle AEM;Mora M;Lynham S;Stirnemann G;Garcia-Manyes S
• 通讯作者: Garcia-Manyes S