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的毁灭性疾病，例如阿尔茨米尔，帕金森氏症，BSE（疯牛病）等。因此，我们需要能够跟踪经历折叠反应的每种蛋白质的折叠途径的实验技术，以确定每个单个蛋白质与“正确”折叠高速公路的何处以及为什么被困在中间状态。现在可以使用最先进的单分子力钳光谱来解决这。使用这种方法，蛋白质通过低（几个piconewton）机械力而展开，一旦力降低，蛋白质就会折叠高度扩展的状态。实际上，我们体内有许多蛋白质在机械力的作用下连续执行其功能。例如，涉及肌肉弹性的蛋白质，在例如心脏组织，必须每天以可逆的方式伸展和放松。如果做到这一点，可能会带来悲惨的后果，导致肌肉萎缩，在最严重的情况下，心脏肌病。因此，了解机械力如何控制这些蛋白质中的蛋白质折叠至关重要，而且远非被理解。为了控制肌肉弹性蛋白弹性，自然设计了内部“锁”，称为二硫键，这阻止了蛋白质在高应力条件下过度拉伸。这种内部机械夹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至关重要。我将使用新型的单分子力钳光谱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展开的蛋白质。该技术已经证明成功地识别了一种蛋白质所采用的不同构象，该蛋白质在进化上旨在折叠生物学时尺度。然而，对于“机械蛋白”所采用的机制几乎不知所措，可以在短时间内且没有能源支出机制在短时间内折叠拉力。我将研究控制肌肉，细胞骨架和细胞外基质中的一系列关键蛋白的构象动力学。接下来，我将研究力对嵌入蛋白质芯中的单个二硫键的减少的影响。特别是，我将研究力如何改变化学反应的结果，并将表征反应的“临界峰值点”的结构，称为过渡态，其中包含有关反应结果的相关化学信息。最后，我将研究二硫键如何影响单个蛋白质的折叠，这种现象在体内发生到组成细胞外基质的多种蛋白质。总的来说，这些单分子技术现在已经达到了一定程度的成熟度，可以将它们用于攻击生物学中更大的挑战，例如导致蛋白质蛋白质和错误折叠的基本生物学机制，尤其是在这些蛋白质中，在这些蛋白质中，保留机械的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

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.

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

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