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

Northwestern University, Evanston IL

Investigators

Abstract

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.