ClpXP is essential for the survival of Escherichia coli ( E.coli) cells mediating the degradation of a number of intracellular proteins. Regulated degradation of specific proteins at the appropriate time is a major task for ClpXP. An understanding of the mechanisms that regulate these appropriately timed events will help to determine and control the outcome of many cellular functions. The AAA⁺ ATPase ClpX subunit of the ClpXP complex is responsible for substrate selection, binding to specific ClpX recognition motifs within the substrate and denaturing the substrate and translocating it to the ClpP proteolytic chamber for subsequent degradation.

Bacteriophage Mu repressor (Rep) is a ClpXP substrate; yet, in its native conformation Rep is relatively resistant to degradation by ClpXP. The final five residues of Rep (CTD5 or VKKAV) serve as a ClpX recognition motif, in that it is able to promote degradation of heterologous proteins although its ability to do so is highly dependent upon its presentation. Furthermore, dominant-negative forms of the repressor, such as the mutant Vir repressors, which have altered C-terminal domains (CTD), not only are rapidly degraded by the ClpXP protease but also trans-target Rep for degradation.

This thesis explores the biophysical dynamics that regulate ClpX selection or recognition of Mu repressors. My results determined that the ClpX recognition motif of Rep is an activatable one, able to undergo activation via conformational changes. Biophysical analyses demonstrate that repressor molecules which acquire exposure and local flexibility of their CTD5 are activated and exhibit a sensitivity to degradation by ClpXP. However, I demonstrate that a constitutively active ClpX recognition motif does not need to be presented on an exposed, flexible segment of the substrate to promote its activation. Finally, I present a novel mechanism for substrate recognition by ClpX. The currently known mechanism for trans-targeting substrates to ClpXP involves tethering the substrate to ClpX by way of an adaptor protein. The results presented here are the first to demonstrate that trans-targeting can occur as a result of conformational changes which promote activation of a ClpX recognition motif located on the substrate.