The nanomechanics of a single protein

单一蛋白质的纳米力学

• 批准号: EP/K00641X/1
• 负责人: Sergi Garcia-Manyes
• 金额: $ 120.02万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FK00641X%2F1
• 关键词: nanomechanics; single; 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, BSE (Mad Cow Disease) and many others. Therefore, we need experimental techniques able to track the folding routes of each individual protein undergoing a folding reaction to identify where and why each individual protein deviates from the 'correct' folding highway, being trapped at an intermediate state. This can be now be addressed by using state-of-the-art single molecule force-clamp spectroscopy. Using this approach, proteins are unfolded by the presence of a low (a few piconewtons) mechanical force, and once the force is reduced, the protein folds from highly extended states. Indeed, there are many proteins in our body that are continuously performing their function under the effect of a mechanical force. For example, the proteins involved in muscle elasticity, with crucial function also in e.g. the heart tissue, have to stretch and relax in a reversible way thousands of time every day. Failing to do that might have tragic consequences, resulting in muscle atrophy and, in the most severe cases, cardiac myopathies. Therefore, understanding how a mechanical force controls protein folding in these proteins is of capital importance, and it is far from being understood. In order to control muscle elasticity protein elasticity, nature has devised internal 'locks', called disulfide bonds, which prevent the protein to overstretch under high stress conditions. Such internal mechanical clamps can be mechanically 'open' through a covalent chemical reaction when required. Therefore, understanding the mechanisms to control these 'mechanical switches' is also of paramount importance in biophysics. I will use the novel single molecule force-clamp spectroscopy technique 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 evolutionarily designed to fold within biological timescales. However, little is known about the mechanisms employed by 'mechanical proteins' to reversibly fold against a pulling force on a short timescale and without the intervention of energy spending mechanisms. I will investigate the conformational dynamics of a series of key proteins that control elasticity in the muscle, in the cytoskeleton and in the extracellular matrix. Next, I will study the effect of force on the reduction of a single disulfide bond embedded within the protein core. In particular, I will study how forces changes the outcome of a chemical reaction, and I will characterize the structure of the 'critical summit point' of the reaction, called transition state, which contains the relevant chemical information on the reaction outcome. Finally, I will examine how disulfide bonds affect the folding of a single protein, a phenomenon occurring in vivo to a wide variety of proteins composing the extracellular matrix. 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 protein and misfolding, especially in these proteins where preserving mechanical extensibility is key to maintain their physiological function.

我们身体的每个器官都是由大量的单个细胞以协调的方式共同工作组成的。在每个细胞内，都有成千上万种不同的蛋白质以一种非常完善和同步的方式执行它们的功能。一般来说，每一种蛋白质都有两种不同的形状——折叠状态和未折叠状态。一旦蛋白质在核糖体中表达出来，它们就会在我们体内不断地展开和折叠，核糖体是制造蛋白质的小工厂。大多数蛋白质只有在它们处于折叠状态时才具有“活性”或“功能”。不折叠会导致无数毁灭性的疾病，如阿尔茨海默氏症、帕金森氏症、疯牛病等。因此，我们需要实验技术来追踪每一个单独的蛋白质进行折叠反应的折叠路径，以确定在哪里以及为什么每个单独的蛋白质偏离了“正确的”折叠路径，被困在中间状态。现在可以通过使用最先进的单分子力钳光谱来解决这个问题。使用这种方法，蛋白质通过低（几皮牛顿）的机械力展开，一旦力减少，蛋白质就会从高度扩展的状态折叠起来。事实上，我们体内有许多蛋白质在机械力的作用下持续发挥着它们的功能。例如，与肌肉弹性有关的蛋白质，在心脏组织中也起着至关重要的作用，每天必须以可逆的方式伸展和放松数千次。如果做不到这一点，可能会产生悲剧性的后果，导致肌肉萎缩，在最严重的情况下，还会导致心肌病。因此，了解机械力如何控制这些蛋白质中的蛋白质折叠是非常重要的，而且还远远没有被理解。为了控制肌肉的弹性，大自然设计了内部“锁”，称为二硫键，防止蛋白质在高压力条件下过度拉伸。这种内部机械钳可以在需要时通过共价化学反应机械地“打开”。因此，了解控制这些“机械开关”的机制在生物物理学中也是至关重要的。我将使用新颖的单分子力钳光谱技术来研究未折叠蛋白质在其原生状态的旅程中所遵循的不同轨迹。这项技术已经被证明是成功的，它第一次识别了一种蛋白质所采用的不同构象，这种蛋白质在进化上被设计成在生物时间尺度内折叠。然而，在没有能量消耗机制的干预下，“机械蛋白”在短时间内对拉力进行可逆折叠的机制知之甚少。我将研究一系列控制肌肉、细胞骨架和细胞外基质弹性的关键蛋白质的构象动力学。接下来，我将研究力对嵌入蛋白质核心内的单个二硫键的还原的影响。特别是，我将研究力如何改变化学反应的结果，我将描述反应的“临界顶点”的结构，称为过渡态，它包含了与反应结果相关的化学信息。最后，我将研究二硫键如何影响单个蛋白质的折叠，这是一种在体内对构成细胞外基质的多种蛋白质发生的现象。总之，这些单分子技术现在已经达到了成熟的水平，它们可以用来攻击生物学中更重大的挑战，例如导致蛋白质蛋白质和错误折叠的基本生物学机制，特别是在这些蛋白质中，保持机械可扩展性是维持其生理功能的关键。

项目成果

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

Forcing the reversibility of a mechanochemical reaction.

• DOI: 10.1038/s41467-018-05115-6
• 发表时间: 2018-08-08
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Beedle AEM;Mora M;Davis CT;Snijders AP;Stirnemann G;Garcia-Manyes S
• 通讯作者: Garcia-Manyes S

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding.

• DOI: 10.1038/ncomms12490
• 发表时间: 2016-08-22
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Beedle AE;Lynham S;Garcia-Manyes S
• 通讯作者: Garcia-Manyes S

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

The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins.

• DOI: 10.1038/ncomms8894
• 发表时间: 2015-08-03
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Beedle AEM;Lezamiz A;Stirnemann G;Garcia-Manyes S
• 通讯作者: Garcia-Manyes S