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明确的三维结构。蛋白质在一段时间内保持这种状态的概率与其热力学稳定性有关：稳定性越高，折叠的活性蛋白质的比例越大。蛋白质在细胞中具有许多不同的功能，包括能量产生、运动和结构作用。在许多这些活动中，可以使蛋白质变性的机械力被施加到这些蛋白质上，并且为了维持它们的功能，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，现在可以在实验室中测量蛋白质的机械强度。这项技术使用一种称为原子力显微镜（AFM）的仪器。然而，已经发现蛋白质的机械稳定性与蛋白质的热力学稳定性无关，而是与蛋白质的不同类型的子结构在每个蛋白质中的排列方式很好地相关。其原因尚不清楚，但据认为，蛋白质中与结构其余部分结合不太紧密的区域可能更有可能在对蛋白质的这一部分施加力时展开。因此，蛋白质展开的力取决于力施加在蛋白质上的点。最近发现，细胞具有大的圆柱形蛋白质复合物，其能够解折叠和消化已被标记用于破坏的蛋白质，以控制细胞过程。据认为，这些“解折叠酶”（例如ClpXP）通过对待降解的蛋白质施加力来解折叠甚至非常稳定的蛋白质。蛋白质被Clp系统解折叠的速率似乎与降解标签位置的蛋白质局部的稳定性相关-观察结果类似于AFM测量的解折叠过程的报告。这两个过程的基本机制目前尚不清楚。该项目旨在通过测量蛋白质中局部不稳定区域的存在如何与蛋白质在使用AFM变性时以及在被细胞解折叠酶ClpXP降解时的机械强度相关来探索蛋白质机械性质的根本起源。

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.