Novel mass spectrometry strategies for the characterization of membrane-bound and oxidative-labeled proteins

Date: 17 January 2020

Venue: UAntwerp, Campus Drie Eiken, Building O, Auditorium O5 - Universiteitsplein 1 - 2610 Wilrijk (Antwerp) (route: UAntwerpen, Campus Drie Eiken)

Time: 4:00 PM - 6:00 PM

PhD candidate: Dietmar Hammerschmid

Principal investigator: Sylvia Dewilde, Frank Sobott

Short description: PhD defence Dietmar Hammerschmid - Department of Biomedical Sciences


In this thesis, I utilized native and structural MS as key methodologies to target structural aspects of the globin-coupled sensor from Geobacter sulfurreducens (GsGCS), the receptor for advanced glycation endproducts (RAGE), and human cytoglobin (CYGB). Both GsGCS and RAGE are membrane proteins, and all endeavors to target the full-length protein, including the membrane domain, by high-resolution techniques, e.g. X-ray crystallography, have not been satisfying yet. The function of CYGB is still under debate, however, evidence is growing that it might have a protective role against oxidative stress. Hence, I studied structural effects of cold atmospheric plasma (CAP) – a technique capable of producing reactive oxygen and nitrogen species (RONS) – on CYGB.

For both GsGCS and RAGE it became possible for the first time to obtain structural data on the full-length protein. In comparison with the globin domain (GsGCS162), GsGCS highlighted a membrane-domain driven oligomerization into an equilibrium between trimers and tetramers, and, more intriguing, a much higher kinetic stability of the ferrous (Fe2+) compared to the ferric (Fe3+) form of GsGCS, which was not observed for GsGCS162. The fact that the higher stability was only observed for GsGCS indicates an oxidation-state dependent helix rearrangement, i.e. (in)activation of a proton transport, for example, of the membrane domain. RAGE has a relatively complex domain architecture with one V- and two C-type domains (akin to Ig domains) on the extracellular side, a transmembrane helix, and a cytoplasmic tail. Therefore, unsurprisingly, the characterization of RAGE showed different oligomerization states depending on which protein construct was analyzed. The full-length and the C2_TM_CT construct exhibited an oligomerization of up to tetramers and hexamers, respectively. These higher oligomerization tendencies were only observed in the presence of the membrane domain. Moreover, collision-induced unfolding experiments, i.e. stability assays, performed on V_C1, V_C1_C2, and FL_RAGE showed a significantly higher stability of V_C1_C2 and FL_RAGE as compared to V_C1, suggesting the C2, which can form a disulfide-mediated dimer, and the transmembrane domain as crucial for the protein’s stability.

Upon oxidative plasma treatment of CYGB, I could identify three major chemical modifications in the protein. Beside the induction of a number of various oxidations, the covalent dimerization via disulfide bridge formation and the binding of NO2 to the heme group could be detected as well.

Overall, native and structural MS have proven as reliable biophysical tools particularly valuable when high-resolution techniques such as X-ray crystallography and/or cryo-electron microscopy lead to unsatisfying results.