How are proteins mechanically unfolded? A study spanning fundamental principles and biological complexity

为了发挥功能，大多数蛋白质需要折叠成一个明确的三维结构。蛋白质在一段时间内保持这种状态的可能性与其热力学稳定性有关：稳定性越大，折叠的活性蛋白质所占的比例就越大。蛋白质在细胞中具有许多不同的功能，包括能量生成、运动和结构作用。在这些活动中，可以使蛋白质变性的机械力被施加到这些蛋白质上，为了保持它们的功能，蛋白质必须具有机械抗性。使用允许操纵单个蛋白质分子的技术，现在可以在实验室测量蛋白质的机械强度。这项技术使用了一种名为原子力显微镜(AFM)的仪器。然而，蛋白质的机械稳定性已被发现与蛋白质的热力学稳定性无关，但与蛋白质中不同类型的亚结构在每种蛋白质中的排列方式密切相关。原因尚不清楚，但人们认为，当用力作用于蛋白质的这一部分时，蛋白质中与结构其余部分结合得不那么紧密的区域可能更有可能展开。因此，蛋白质展开的力取决于施加在蛋白质上的力的点。最近发现，细胞具有大的圆柱形蛋白质复合体，能够展开和消化标记为要销毁的蛋白质，以控制细胞过程。据认为，这些“去折叠酶”(例如ClpXP)通过对被降解的蛋白质施加压力来展开甚至非常稳定的蛋白质。CLP系统解开蛋白质的速度似乎与降解标签位置上蛋白质局部的稳定性有关--这一观察结果类似于AFM测量的解折叠过程的报道。目前，这两个过程的基本机制都是未知的。该项目旨在通过测量蛋白质中局部不稳定区域的存在与蛋白质在使用原子力显微镜变性和被细胞展开酶ClpXP降解时的机械强度之间的关系，来探索蛋白质力学性质的根本来源。

To be functional most proteins need to fold up into a well defined three dimensional structure. The probability that a protein remains in this state over a period of time is related to its thermodynamic stability: the greater the stability, the greater the proportion of folded active proteins. Proteins have many different functions in cells including energy generation, locomotion and structural roles. Mechanical force, which can denature proteins, is applied onto these proteins during many of these activities and, to maintain their function, proteins have to be mechanically resistant. Using techniques that allow the manipulation of single protein molecules, it is now possible to measure the mechanical strength of proteins in the laboratory. This technique uses an instrument called the atomic force microscope (AFM). However, the mechanical stability of proteins has been found to be unrelated to a protein's thermodynamic stability but correlates well with how the different types of a protein's sub-structure are arranged in each protein. The reason for this is unclear but it is thought that regions in proteins that are bound less tightly to the rest of the structure may be more likely to unfold when force is applied onto this part of the protein. As a consequence, the force at which a protein unfolds depends upon the points at which the force is exerted onto the protein. It has recently been discovered that cells possess large cylindrical protein complexes that are able to unfold and digest proteins which have been tagged for destruction in order to control cellular processes. It is thought that these 'unfoldases' (ClpXP for example) unfold even very stable proteins by applying force onto the proteins to be degraded. The rate at which proteins are unfolded by the Clp system appears to correlate with the stability of the protein local to the position of the degradation tag - an observation similar to that reported for the unfolding process measured by the AFM. The underlying mechanism for either process is, at present, unknown. This project aims to probe the fundamental origins of the mechanical properties of proteins by measuring how the presence of regions of local instability in proteins correlates with the mechanical strength of proteins when denatured using the AFM and when degraded by the cellular unfoldase ClpXP.