This result was expected since intramolecular binding of Mpa subunits is very tight; the complex fails to dissociate on a chromatographic sizing column. Pup-GGQ binds to Msm Mpa primarily via the Cterminal half of the helical region. In addition, a short region of the N-terminus, T6-R8, and C-terminus, E52-Q64, are affected by Msm Mpa binding. Titrating Msm Mpa into Pup-GGQ in vitro results in a gradual uniform broadening of the peaks from residues S21 to Q60, excluding the C-terminal residues, K61, G62, G63 and Q64. In-cell, peak intensities do not decrease for all the residues in the Pup-GGQ helix, S21A51. We suggest that this difference is due to the interactions of Pup-GGQ with components of cytosol that block part of the interaction surface between Pup and Mpa observed in vitro. The in-cell observations are also in general agreement with specific contacts in the helical region, identified in the Pup-Mpa co-crystal, although in this instance, truncated Mpa was used. The use of a truncated Mpa may not accurately represent the Pup-Mpa interaction due to the possibility of altered conformations resulting from the truncation and from in vitro conditions that fail to duplicate the cellular environment in which this interaction normally occurs. In cells expressing Pup-GGQ, Msm Mpa, and WT or Opengate proteasome CPs, the intracellular concentration of CP is significantly less than that of Msm Mpa and Pup-GGQ. In this case, the Pup-GGQ/Msm Mpa complex will be the predominant species. Nevertheless, the in-cell NMR spectrum of the Pup-GGQ/Msm Mpa complex in cells expressing non-stoichiometric amounts of proteasome CPs is different from that of cells expressing only Pup-GGQ and Msm Mpa: the presence of proteasome CPs results in the complete broadening of peaks associated with the N-terminal tail of Pup-GGQ. The Pup-GGQ/Msm Mpa complex appears to be stabilized by non-stoichiometric amounts of proteasome CPs. Since the proteasome CP binds to Msm Mpa with low affinity, we postulate that transient binding of the proteasome CP to the Pup-GGQ/Msm Mpa complex results in the N-terminal tail of Pup-GGQ being occluded by the Msm Mpa central cavity, which leads to complete broadening of the Pup-GGQ spectrum. Different in-cell BAY 73-4506 spectra result when the order of overexpression of Pup-GGQ and the Msm Mpa/proteasome CP complex are reversed. The Msm Mpa/ proteasome CP complex can take up to 12 hours to fully assemble in the cell following induction of over-expression. When Pup-GGQ is over-expressed first, the result is a mixed population of cells containing free Pup-GGQ, Pup-GGQ bound to Msm Mpa and Pup-GGQ bound to the Msm Mpa/ proteasome complex. The corresponding in-cell NMR spectrum will represent an average of these three spectra. In this case, the spectrum is very similar to that of Pup-GGQ in complex with Msm Mpa, reflecting the interaction between Msm Mpa and the helical region of Pup-GGQ. When the Msm Mpa/proteasome CP complex is over-expressed first, the in-cell spectrum is completely broadened. Nutlin-3 Western analysis demonstrated that this is not due to degradation of Pup-GGQ. Unlike Ubiquitin, which is recycled in the eukaryotic proteasome, both free and target-bound Pup-GGE are degraded in the mycobacterium proteasome, but may be recycled by depupylase/deamidase Dop. Pup-GGQ is a precursor molecule that is converted to Pup-GGE before being ligated to a substrate targeted for degradation. Unlike Pup-GGE, which is a very poor substrate, Pup-GGQ is a not a substrate for proteasomal degradation, consistent with its function as a precursor molecule. Indeed, free Pup-GGQ can be detected in Dop mutants of both M. smegmatis and M. tuberculosis strains, albeit in low concentration. STINTNMR experiments present a dynamic picture of the fate of PupGGQ inside a cell containing Mpa and proteasome CPs.