The nanomechanics of a single protein

我们身体中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，有成千上万种不同的蛋白质，它们以一种非常完善和同步的方式发挥作用。一般来说，这些蛋白质中的每一种都可以以两种不同的形状存在-折叠和未折叠状态。一旦蛋白质在核糖体中表达，它们就会在我们的身体中不断地展开和重新折叠，核糖体是生产它们的小工厂。大多数蛋白质只有在处于折叠状态时才具有“活性”或“功能”。不折叠会引起无数毁灭性的疾病，如阿尔茨海默氏症，帕金森氏症，疯牛病（疯牛病）和许多其他疾病。因此，我们需要实验技术来跟踪每个蛋白质的折叠路线，以确定每个蛋白质在哪里以及为什么偏离“正确”的折叠高速公路，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（几微微牛顿）机械力的存在而展开，一旦力减小，蛋白质就会从高度伸展状态折叠。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其功能。例如，与肌肉弹性有关的蛋白质，在心脏组织中也具有重要功能，每天必须以可逆的方式拉伸和放松数千次。如果做不到这一点，可能会产生悲惨的后果，导致肌肉萎缩，在最严重的情况下，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，而且还远未被理解。为了控制肌肉弹性蛋白质弹性，大自然设计了内部“锁”，称为二硫键，防止蛋白质在高应力条件下过度拉伸。当需要时，这种内部机械夹具可以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。我将使用新的单分子力钳光谱技术来研究不同的轨迹所遵循的未折叠的蛋白质在其旅程的天然状态。这种技术已经被证明是成功的，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，很少有人知道的机制所采用的“机械蛋白质”可逆地折叠对拉力在短时间内，没有干预的能量消耗机制。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的单个二硫键还原的影响。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，其中包含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在构成细胞外基质的各种蛋白质中。总而言之，这些单分子技术现在已经达到了成熟的水平，它们可以用于攻击生物学中更重要的挑战，例如导致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，保持机械可延伸性是维持其生理功能的关键。

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.