Often, during assembly, effector molecules such as ligands or substrates are also present. The presence of these molecules prior to or following the expression of components of the complex can play a regulatory role in the LDN-193189 assembly of that complex. Furthermore, the binding of these effector molecules may alter the pathway through which proper, biologically active conformations are 
achieved. It is not clear a priori that the final conformation and commensurate activity of the complex will be different due to this temporal control. One method to study macromolecular complexes inside a cell that affords temporal control over assembly is STINT-NMR. STINT-NMR is used to Torin 1 elucidate STructural INTerations between proteins within their native environment by using incell NMR. In STINT-NMR, protein over-expression is induced in labeling medium to produce a uniformly labeled target protein containing NMR-active nuclei; cells are then transferred to non-labeling medium to induce overexpression of the interactor protein. Most importantly, the order of sequential over-expression of target and interactor proteins can be reversed, allowing temporal control over the assembly of the complex. In this work STINT-NMR was used to examine the interactions between the Ubiquitin-like protein, Pup, in the presence of the Mycobacterium proteasome ATPase, Mpa, and the active 1.2 megadalton proteasome complex, consisting of Mpa and the Mycobacterial proteasome core particle. The importance of this macromolecular complex is underlined by the fact that Mycobacterium tuberculosis is particularly resistant to reactive nitrogen intermediates generated by host immune system, and this resistance is related to the proteasome and mpa. The Mtb proteasome CP consists of 14 copies each of two distinct but related polypeptides, �� and ��. The overall architecture of the CP is conserved: ��- and ��-type subunits segregate into four homo-oligomeric 7-member rings. Two juxtaposed rings of ��-type subunits are flanked on top and bottom by a ring of ��-type subunits to form the barrel-shaped complex. The main function of the ��-rings is to form a gated channel that controls the passage of unfolded substrates into and cleaved particles out of the proteolytic chamber. Studies have shown that Mtb Mpa forms 404 kDa hexameric rings similar to AAA+ ATPases found in the eukaryotes. Structural analysis predicts that Mtb Mpa physically interacts with the ��-rings of Mtb proteasome CP and plays a role in binding, unfolding and translocating substrates into the proteasome complex. Proteins that are targeted for degradation in eukaryotes are generally tagged with the Ubiquitin, a small highly conserved regulatory protein. By using Mtb Mpa as bait in a bacterial two-hybrid screen of an Mtb genomic library searching for potential binding partners of Mtb Mpa, the first prokaryotic Ubiquitin-like protein, Pup was discovered. Pup is a 64 amino acid protein that modifies and targets mycobacterium proteins to the proteasome for degradation. Pup is similar in size to Ubiquitin but the two proteins have different sequences and lack structural homology. Pupylated proteins, which have been tagged with Pup, interact with Mtb Mpa. The Mtb proteasome complex presents a tractable in-cell system for studying the interactions between Pup and the proteasomal ATPase, Mpa. Crystal structures of the Mtb proteasome CP, the Pup-Mpa coiled coil domain complex, as well as in vitro NMR solution studies of PupMpa interactions are available. E. coli is a relevant prokaryotic host that provides a proper milieu for studying the Pup-Mpa interaction without interfering factors. Indeed, Mtb Pup ligase, pafA, was overexpressed in E. coli to study pupylation of proteins and to prove that no additional factors are required for this process. Individual ��- and ��-subunits of the Mtb proteasome core particle, also called prcA and prcB.