Affinity-based protein profiling (AfBPP) using different Callyaerin probes in M. tuberculosis H37Rv

The natural cyclopeptide Callyaerin B (CalB) exhibits highly specific antitubercular activity. The aim of this project is to study the protein binding partner of this compound using either activity-based protein profiling (ABPP) (ACE_0721) or affinity-based protein profiling (AfBPP) (ACE_0682 & ACE_0721). To enable a two-step enrichment protocol, CalB probes modified with an alkyne group (CalB_C4Pra) and optionally additional photoleucine (CalB_R5phLeu) or 4-benzoyl-phenylalanine (CalB_R3Bpa) as photoactive groups were used. For this purpose, M. tuberculosis H37Rv cells were treated with the respective probe for three hours at 37 °C and then exposed to UV light (λ = 365 nM) for 20 minutes at room temperature. The cells were lysed, and biotin was attached to probe-labeled proteins using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). These proteins were subsequently enriched with an avidin-matrix, which was thoroughly washed with a 1% SDS solution. Furthermore, a competitive AfBPP approach was chosen, to distinguish specific protein labeling from background labeling (ACE_0721). For this, the cells were treated with excess of unmodified CalB for 30 minutes prior to the addition of the respective photoprobe. In addition, it has been shown, that the susceptibility of M. tuberculosis H37Rv to CalB is highly dependent on the expression of the putative membrane protein Rv2113. A gene deletion mutation of this protein (Δrv2113) was used to identify further unspecific labeling of the AfBPP approach (ACE_0740).