Date of Award
Master of Science
Dr. Lars Konermann
Much is known regarding the folding reactions of small, single domain proteins. However, the extent to which these principles apply to the folding of large, multi-domain proteins, and multi-subunit protein complexes, remains to be seen. Monitoring the (un)folding reactions of such proteins poses unique challenges, and requires instrumentation capable of distinguishing intramolecular contacts from intermoleculer interactions.
Electrospray ionization (ESI) mass spectrometry (MS) offers enormous potential to the study of multi-subunit protein complexes, as it simultaneously detects and resolves coexisting protein species and conformers in solution. Such high selectivity combined with excellent sensitivity makes ESI-MS a primary tool in the study of higher-order protein folding reactions.
The work herein examines the unfolding reaction of the multi-subunit oxygen transport protein hemoglobin (Hb) using ESI-MS in combination with UV-Vis spectroscopy. The results strongly support a symmetrical dissociation mechanism, which contradicts a previously proposed mechanism supporting an asymmetric disassembly. This apparent contradiction is attributed to non-native oxidative modifications which destabilize the native state.
Due to the nature of ESI, instrument-induced analyte oxidation is a concern. The process of ESI-induced analyte oxidation is discussed and the mechanism of which is determined. Prevention strategies are therefore put forth, since given the effects of
oxidation on folding, it is vital to distinguish solution phase analyte oxidation from instrument-induced oxidation.
Boys, Brian L., "EFFECTS OF OXIDATIVE MODIFICATIONS ON SUBUNIT INTERACTIONS IN HEMOGLOBIN" (2009). Digitized Theses. 4029.