SGER: Exploration of the Mechanism of ATP-dependent Proteases by Force Measurements Using Single Molecule Techniques

蛋白质在细胞中的展开是几个过程中的一个重要步骤，最明显的是蛋白质在一些膜上的转运和ATP依赖的蛋白酶对蛋白质的降解。线粒体蛋白转位酶和ATP依赖的蛋白水解酶通过依次分解它们的底物来催化去折叠。底物蛋白质对展开的敏感性的差异似乎对易位和降解的特异性有很大贡献。这里要检验的新假设是，线粒体和依赖于ATP的蛋白酶通过物理上拉动多肽链来催化蛋白质的展开。通过拉力展开将是细胞过程的一种新的作用机制，并将对理解许多分子机器产生重要的影响。拉动机制将与实验观察一致，但该图像是基于与宏观世界中的经验的类比，目前还不清楚该图像是否可以转移到正在讨论的分子事件中。现在，至少在原则上，通过实验来解决这些问题是可能的。在过去的五年里，已经产生了使用原子力显微镜和光学陷阱来测量生物机器产生的微小物理力的方法。这个项目的长期目标是解决以下三个广泛的目标：1)确定一种依赖于ATP的蛋白酶ClpAP是否产生物理拉力。拉力及其对底物的影响将通过测量蛋白酶可以产生的最大力、移位率和产生拉力的马达的步长来表征。2)确定拉力是否以及如何取决于衬底的顺序。初步证据表明，底物中的低复杂性区域，如谷氨酰胺重复区域和甘氨酸重复区域，会减弱蛋白酶体的去折叠活性。这里的假设是，重复序列影响蛋白酶中的易位马达与底物相互作用的方式。3)比较不同的蛋白酶产生的力，并确定哪些属性是所有蛋白酶固有的，哪些属性是不同的。该SGER项目的直接目标是为上述实验建立生化基础。将蛋白水解酶ClpAP偶联到聚苯乙烯小球上，保证了酶的活性。将构建一个合适的蛋白酶底物。它应该足够长，以便能够监测降解过程中衬底的力和位移。底物将被交联到聚苯乙烯珠子上，并确定底物是否仍可被蛋白酶访问。一旦完成，该系统将准备好使用原子力显微镜来确定蛋白质展开过程中的物理力。广泛的影响：在这个高风险的项目中要探索的概念是新的。如果这些概念被这项工作证明是正确的，那么一种前所未有的生物机器的行动机制将被建立起来。这将对分子机器在纳米生物技术应用中的使用产生影响。该项目还将涉及学生培训活动。

Protein unfolding in the cell is an important step in several processes, most clearly protein translocation across some membranes and protein degradation by ATP-dependent proteases. The mitochondrial protein translocase and ATP-dependent proteases catalyze unfolding by sequentially unraveling their substrates. Differences in the susceptibility of substrate proteins to unfolding appear to contribute significantly to the specificity of translocation and degradation. The novel hypothesis to be tested here is that mitochondria and ATP-dependent proteases catalyze protein unfolding by physically pulling at the polypeptide chain. Unfolding by pulling would be a new mechanism of action for a cellular process and would have important consequences for understanding of many molecular machines. A pulling mechanism would be consistent with the experimental observations but the image is based on analogies to the experiences in the macroscopic world and it is not immediately clear whether this image can be transferred to the molecular events under discussion. It is now possible, at least in principle, to address these questions experimentally. The last five years have produced the methodology to measure small physical forces produced by biological machines using atomic force microscopy and optical traps. The long term goals of this project are to address the following three broad objectives:1) To establish for one ATP-dependent protease, ClpAP, whether it produces a physical pulling force. The pulling force and its effect on the substrate will be characterized by measuring the maximum amount of force that the protease can generate, the translocation rates, and the step size of the motor that generates the force. 2) To determine whether and how the pulling forces depend on the sequence of the substrate. Preliminary evidence suggests that low complexity regions, such as glutamine repeat regions and glycine repeat regions in substrates attenuate the unfolding activity of the proteasome. The hypothesis here is that the repeat sequences affect the manner in which the translocation motor in the protease interacts with the substrate.3) To compare the forces produced by the different proteases and to determine what properties are intrinsic to all proteases and what properties vary. The immediate goal of this SGER project is to setup the biochemical groundwork for the experiments described above. The protease ClpAP will be cross-linked to polystyrene beads and activity of the protease will be ensured. A suitable protease substrate will be constructed. It should be long enough to allow monitoring of the forces and displacement of the substrate during degradation. The substrate will be cross-linked to the polystyrene beads and determine whether the substrate remains accessible to protease. Once completed, the system will be ready for use in determination of physical forces during protein unfolding using atomic force microscopy.Broader impacts: The concepts to be explored in this high-risk project are novel. If the concepts were proved correct by this work, an unprecedented mechanism for the action of a biological machine will be established. This will have consequences in the use of molecular machines in nanobiotechnology applications. The project will also involve student training activities.