Researcher(s)
- Yuehui (Emily) Lu, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Karl Schmitz, Department of Biology, University of Delaware
- Farzaneh Hojjati, Department of Biochemistry, University of Delaware
Abstract
The ATP-dependent ClpC1P1P2 protease is an essential protein quality-control complex in Mycobacterium tuberculosis and represents a promising target for the development of new antibacterial therapies. Recent studies suggest that phosphoarginine (pArg) modifications can serve as degradation signals recognized by ClpC1, the unfoldase component of the protease. However, the mechanisms governing pArg-dependent substrate recognition by ClpC1 remain poorly understood. This project aims to develop and optimize phosphoarginine-bearing model substrates that can be used to investigate ClpC1-mediated proteolysis and substrate recognition.
To support these studies, recombinant protein constructs were cloned, expressed in Escherichia coli, and purified. Purification workflows for His-tagged and Strep-tagged proteins were established using Ni-NTA affinity chromatography, TEV protease cleavage, and Strep-Tactin affinity chromatography. The Paenibacillus glacialis arginine kinase McsB (PglMcsB) and a fluorescent model substrate (I27-GFP) were successfully expressed and purified for downstream phosphorylation experiments. PglMcsB was used to phosphorylate arginine residues on substrate proteins, while I27 is a model substrate with no native arginine residues on which arginines were introduced through site-directed mutagenesis, allowing us to control Arg number and position. These purified proteins will be used in phosphorylation reactions, followed by ATPase and proteolysis assays to identify conditions that generate functionally active phosphoarginine-bearing substrates and promote their degradation by ClpC1P1P2.
Although phosphorylation assays and optimization are currently in progress, establishing reliable purification methods provides an important foundation for future experiments. This established workflow will facilitate the production of high quality phosphoarginine bearing substrates for investigating ClpC1 substrate recognition and proteolysis. These studies will contribute to a better understanding of ClpC1 function in M. tuberculosis and may support future efforts to develop ClpC1-targeted antibacterial strategies.