Development of hydrogen deuterium exchange mass spectrometry methodologies for the analysis of bacterial nano-compartments
Lambert, Thomas Ole Tandrup
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a versatile biophysical technique used in a variety of applications within structural proteomics. The key advantages of HDX-MS are that it can provide insight into conformational changes in protein structure as well as allowing investigation of the details of protein-protein interactions, protein-small molecule interactions and protein folding. For these reasons it is an often-used, complementary technique when paired with other biomolecular techniques such as X-ray crystallography, cryogenic electron microscopy (Cryo-EM) and native mass spectrometry. Encapsulin nano compartments (Enc) or encapsulins are a family of bacterial proteins which form large icosahedral protein cages. These protein cages are of a large enough size (25-30 nm) to allow for the encapsulation of smaller cargo proteins. It has been found that compartmentalization plays an important physiological role within bacteria. One recently discovered ENC, encapsulated ferritin (EncFtn), is known to act as an iron storage system. Although details of Enc-EncFtn structure are beginning to emerge, several questions remain regarding dynamics and interactions of this complex. This thesis focuses on the application of HDX-MS for the study of these important bacterial nano-compartments. This body of work initially explores the assembly pathway of EncFtn derived from Haliangium ochraceum using a bottom-up HDX-MS workflow. It was previously shown that EncFtns characteristically exist as a decameric ‘pentamer-of-dimers’ arrangement. HDX was used to determine the details of the assembly pathway and the formation of the decameric assembly via recruitment of specific dimer subunits. This study’s second aim was to analyse the structures of both a loaded encapsulin (Enc-Loaded) and empty encapsulin (Enc-Empty). Differential HDX-MS was used to reveal significant differences in deuteration exchange 6 between both the empty and loaded forms. The regions of higher deuterium uptake in Empty encapsulin were localized within the interior of the encapsulin and surrounded a hypothesised cargo binding sequence that been previously determined via Cryo-EM. This HDX analysis also highlighted the dynamic and flexible nature the putative entry pore within the ENC. Finally, a novel automated HDX method was developed for top-down HDX analysis of proteins. This strategy allows for single residue resolution of a deuterium uptake event with the potential of proteoform isolation and selection. Fourier Transform Ion Cyclotron Resonance (FT-ICR MS) was coupled with an automated HDX platform and Electron Capture Dissociation (ECD). This workflow was applied to study EncFtns, and direct comparison with traditional bottom-up data demonstrated the feasibility and potential advantages of this new workflow.