Which proceeds through autocatalytic removal of an N-terminal peptide. Pup contains a di-glycine motif at the penultimate position of the C-terminus, followed by either glutamate or glutamine, depending on the organism. Mass spectrometry revealed that for pupylated substrates in Mtb, the C-terminal Gln is not removed, but rather deamidated to Glu prior to being conjugated to substrate lysines. Here we present how Pup-GGQ interacts with the Mpa-proteasome complex inside a cell by recreating the final steps in the mycobacterium degradation pathway in E. coli. We used PupGGQ for our studies since it exists as a free molecule inside the cell, not VE-822 attached to its target. Pup residues 21 through 51 exhibit a propensity to form a transient ��-helical structure : 13C chemical shifts of ��and ��-carbons in this region of the protein are consistent with partial ordering of the structure, while the lack of dispersion in the amide region of the NMR spectrum suggests a disordered state. The crystal structure of the Pup-Mpa complex shows a helix conformation when Pup-GGE binds to the coiled coil domain of Mpa. The binding induces a stable helical conformation encompassing amino acids 21-51 of Pup-, while the N- and C-termini remain unstructured in the Pup-Mpa complex. To investigate the structural role of the interaction between the ��-helix and Mpa under in-cell conditions, STINT-NMR was used to characterize the interaction surface of Pup-GGQ when bound to Mycobacterium smegmatis Mpa, Msm Mpa. The differential broadening observed in the resulting spectra are characteristic of intermediate exchange and reflect an equilibrium between free and bound Pup-GGQ since Pup-GGQ is in excess and 
Mpa is not over-expressed to a sufficiently high level to form a large population of a complex. In general, the same regions of Pup-GGQ are most strongly perturbed as in the previous experiment, and while the magnitudes of the chemical shift changes are comparable, the magnitudes of the VE-821 intensity changes are reduced, consistent with sub-stoichiometric populations of Pup-GGQ and Mpa. The order of expression appears to have no significant effect on the regions of Pup-GGQ affected by Mpa binding. We conclude that Msm Mpa assembles into the same conformational state regardless of the absence or presence of a high concentration of its physiological ligand. Assembly of macromolecular machinery in the presence of native ligands in the crowded cytosol presents a complicated system for study by amino-acid residue resolution techniques. We used in-cell STINT NMR to map the interactions of the prokaryotic ubiquitin-like protein, Pup, with the mycobacterial proteasome in E. coli. The intracellular medium provides a prokaryotic environment for structural study of Mtb proteasome function without the complications of additional factors that may specifically interact with this system. Reconstructing the interactions between the mycobacterial Msm Mpa/Mtb proteasome CP complex and Pup-GGQ inside a cell at aminoacid residue resolution has allowed us to examine intracellular processes that are not accessible by in vitro investigations. In vitro studies showed that Pup is a disordered protein possessing a transient helical structure in its C terminal region. As in the case of ��-synuclein, physiological conditions result in a seemingly disordered protein may acquire stable secondary and even tertiary structure. Only minor changes in the in-cell NMR spectrum of Pup-GGQ occur when compared to the cell lysate spectrum. This suggests that PupGGQ does not possess a stable secondary structure in the cytosol. Since Pup-GGQ acts as an anchor for the proteasome system, with the N-terminus assuming an extended structure, the disorder may be important for its function.