The overall goal of this project is the elucidation of the mechanism of ATP (adenosine triphosphate-dependent proteolysis by Lon protease. Lon protease is an ATP-dependent serine protease complex functioning to maintain cellular homeostasis and relieve cell stress. This protease possesses an intrinsic ATPase activity that is elevated during protein degradation. The specific goal of this project is to determine how the chemical energy source ATP is used by Lon to catalyze protein degradation, and from here, obtain insights into why nature design proteases that utilize ATP as activators. In this project, the PI will perform kinetic experiments to identify enzyme intermediates, whose formations are dependent on ATP hydrolysis in the degradation pathway of an unstructured protein called lambda N, and determine their functions accordingly. The kinetic mechanisms of ATP binding and subsequent hydrolysis during lambda N degradation are evaluated by rapid chemical quench techniques. The kinetic mechanisms of ATP- versus AMPPNP-dependent cleavage of specific sites within lambda N are evaluated by fluorescence stopped flow technique. The kinetic mechanisms of ATP- versus AMPPNP-dependent translocation of a defined scissile site in lambda N to the proteolytic site of a bacterial Lon are evaluated through monitoring the fluorescence resonance energy transfer signal by stopped flow techniques. Results generated from this study will provide insight into the mechanism by which Lon couples its ATPase and peptidase activities to degrade an endogenous protein substrate lacking defined structure. This work will represent the first quantitative study designed to determine the contribution of the ATPase activity of Lon to protein degradation.
Broader Impacts This project employs a multi-disciplinary approach to determine the mechanism of ATP-dependent proteolysis. As such, students participating in this project acquire a multidisciplinary training in enzyme kinetics, protein chemistry, liquid chromatography, mass spectrometry, chemical synthesis, solid phase peptide synthesis, fluorescence spectroscopy, protein purification and molecular cloning. These research activities provide excellent training opportunities to undergraduate and graduate students in biochemistry. Additionally, all personnel working in the PI's laboratory serve as volunteers to the National Youth Sports Program (NYSP). NYSP is summer program offered to children of low-income families in the Greater Cleveland area between the ages of 10 and 16. Participating students teach these children chemistry of daily life through experimentation with household chemicals and dry ice. This annual outreach activity has become a tradition in the PI's laboratory, where undergraduate and graduate students learn to become more involved in the community and education for the underprivileged.
Project outcomes that address tntellectual merrit: The goal of this study was to determine how the Escherichia coli ATP-dependent protease known as Lon utilizes adenosine triphosphate (ATP) to catalyze the degradation of an endogenous substrate known as the lambda N protein. As the lambda N protein lacks any defined structure, its degradation by Lon serves as a study model for elucidating the timing of ATP usage, delivery of the different scissile sites within lambda N to the proteolytic site of Lon and the cleavage of the different scissile site within an unstructured substrate. To accomplish our goal, we utilized a combined transient kinetics and fluorescence resonance energy transfer approach. A collection of lambda N proteins containing a unique fluorescent label at different positions along the substrate was generated and used to probe the order by which by which each label approached the proteolytic site of Lon and then cleaved. The kinetics for the ATP consumption triggered by lambda N degradation was also independently determined. When complied together, our findings provided a quantitative working model to account for the mechanism of ATP-dependent proteolysis of unstructured substrates. The model will benefit future work in accessing how Lon mutants showing defects in ATP consumption or substrate delivery or peptide bond cleavage step affects the overall performance of the protease. The approach developed for studying Lon should be applicable to studying other ATP-dependent proteases. Broader Impact: The activities supported by this award provide ample training opportunities for Ph.D. graduate and undergraduate students in biochemistry, molecular cloning, protein purification, chemical synthesis, fluorescence spectroscopy and enzyme kinetics. All the students participating in this project published research articles. Undergraduate students who left my lab either continue attended graduate or medical schools. Most of the Ph.D. students supported by this award intended to pursue an academic career. Additionally, all personnel working in the Lee lab served as volunteers to the National Youth Sports Program (NYSP). NYSP is a summer program offered to children of low-income families in the Greater Cleveland area between the ages of 10 and16. The Lee lab teaches the children chemistry of daily life through experimentation with household chemicals and dry ice. This annual outreach activity has become a tradition in the Lee lab, where undergraduate and graduate students learn to become more involved in the community and education for the underprivileged.
