91原创

Clp proteases mechanically unfold and degrade protein substrates through the collaboration of an ATP-dependent unfoldase and a barrel-shaped peptidase. Our research aims to establish how their activity is regulated in the cell, how they select specific substrates from the complex cytoplasmic proteome, and how structural features relate to their proteolytic activity. We use a variety of experimental tools, including biophysical and biochemical assays to probe protease assembly and function, X-ray crystallography to reveal protein structure, and molecular microbiology approaches to interrogate proteolytic processes in the cell. We hope to exploit our understanding of these enzymes to identify novel antibacterial compounds that target Clp proteolysis in聽M. tuberculosis聽and other pathogenic bacteria.

Substrate identification and selectivity

Clp proteases selectively recognize protein substrates, which minimizes wasteful and deleterious off-target proteolysis. However, few聽bona fide聽substrates are known in mycobacteria, and the rules that govern substrate discrimination are not understood. Through targeted screening and capture-based methods, we aim to identify novel physiological substrates. We use cell-based and phage-based screening approaches to define the sequence-based rules by which these proteases select substrates. We also aim to crystallize protease components in complex with substrate polypeptides, to determine the specific interactions and motifs that guide substrate recognition. These studies will help us understand the role that Clp proteases play in mycobacterial biology, and will guide the development of novel聽in vivo聽reporter substrates.

High-throughput screening and compound characterization

A major motivation for studying Clp protease function is to improve our ability to develop compounds that disrupt their activity in聽M. tuberculosis. While we have a rich toolbox of reagents and assays useful for probing protease activity聽in vitro, few of these tools are well-suited to high-throughput screening applications. We are working to develop robust assay platforms with higher signal-to-noise, improved dynamic range, and the ability to multiplex reporters for unfolding, peptidase, and protease activities. We also collaborate with talented synthetic chemists to characterize and optimize compounds that target these essential mycobacterial enzymes.