Optimizing a ClpP trap assay for studying degradation under stressful conditions in E. coli

Abstract

Cells can reshape their collection of proteins by altering either gene expression or proteolysis. However, though gene expression has been carefully characterized during stationary phase, less is known about how protein degradation changes in stationary phase. In bacteria like E. coli, many proteins are degraded by the conserved ClpXP protease, where ClpX recognizes and unfolds substrates, and then channels them into the ClpP chamber for digestion. Previously a ClpPtrap was developed whereby an affinity-tagged ClpP was mutated in its active site (S97A) so that substrates remained trapped in the proteolytic chamber. After purification these substrates can be identified by mass spectrometry. Here we modify and optimize this assay to study how ClpXP modifies protein degradation under stressful conditions such as the entry into stationary phase.