Navigating the oligomeric landscape of the periplasmic stress response protease-chaperone DegP with charge detection mass spectrometry

DegP is a crucial protease-chaperone protein found in Gram-negative bacteria. Within the periplasmic space, this enzyme exists as a dynamic ensemble of oligomers, formed either by trimeric building blocks or through two competing self-assembly pathways. The formation of these oligomers is influenced by the size of bound client proteins, with possible arrangements including 12mers, 24mers, and 60mers.

Previous research methods, such as dynamic light scattering, analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and electron cryomicroscopy, were employed to map the oligomeric landscape of DegP. However, these techniques generally provide ensemble averages and struggle to differentiate closely related coexisting species.

To overcome this limitation, the present study employed charge detection mass spectrometry (CDMS) to directly measure the masses of individual DegP ions. This approach allows for the resolution of the complete oligomeric distribution, both in the absence and presence of four clients varying in size.

The results obtained using CDMS revealed previously unidentified odd-numbered oligomers and provided quantitative data on the relative abundance of each assembly type. Additionally, heat-cool cycling CDMS experiments were conducted to observe changes in cage distribution. These experiments revealed the protective role of DegP in client proteins and the subsequent refolding process. As a result, CDMS proves to be a highly effective single-molecule tool for analyzing heterogeneous protein assembly landscapes.