Two fundamental questions about any motor are: 1) how far does the machine travel per turnover of the engine and 2) how much fuel is required? Answering these two questions is essential for understanding how a motor operates in a car or how a motor operates in the cell of a living organism. However, answering such questions for a molecular motor in the cell, which cannot be directly observed by eye, requires substantially different techniques and experimental strategies. Molecular motors that exist in the cell are essential for a vast array of metabolic processes. For example, survival of extreme heat or exposure to environmental stresses depends upon molecular motors that either repair or remove damaged cellular components. If such damaged components were left unattended they would have catastrophic effects on the cell. At the core of this work the researchers seek to answer the two posed fundamental questions of how two representative motors tasked with such repair activities move along their track and how much energy they require to do so. The knowledge gained will be important for gaining a deeper understanding of how these motors and a vast array of similar motors operate and potentially the impact they have on the cell. Moreover, the results will allow others to better propose and test mechanisms for a variety of motor proteins that use similar operating principles and lie at the heart of a vast array of biological functions. Vital to the research infrastructure of our nation is training the next generation of scientists with the ability to apply the thermodynamic and transient state kinetic approaches proposed here. This goal will be achieved by involving graduate, undergraduate, and high school researchers in the work; a strategy that has been successfully invoked with previous NSF support.
In all organisms reactions such as protein remodeling, ATP dependent proteolysis, and protein disaggregation are essential for proteome maintenance. However, the molecular level events in these reactions remain obscure. This research will advance knowledge by providing a detailed molecular mechanism for ClpA, ClpAP, and ClpB catalyzed polypeptide translocation, which represent model enzymes that catalyze protein remodeling, ATP dependent proteolysis, and protein disaggregation, respectively. In addition, the results will advance knowledge across fields because a variety of homologous AAA+ enzymes are thought to use similar mechanisms to catalyze such disparate reactions as clamp loading in DNA replication, microtubule severing, membrane fusion, and morphogenesis and trafficking of endosomes. This project will promote teaching, training and learning and perform outreach by incorporating high school students and undergraduate researchers through the university's ChemBridge and Chemistry Scholars programs, respectively. A further benefit of the proposed activity to society is that this work will yield individuals highly trained in the application of an array of biophysical and molecular biology techniques.
