Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Dr. Lars Konermann


Membrane proteins continue to represent a major challenge for most analytical techniques. Using bacteriorhodopsin (BR) as model system, this work aims to develop mass spectrometry (MS)-based approaches for exploring the structure, dynamics and folding of membrane proteins.

As the first step, BR in its native lipid environment was exposed to hydroxyl radicals, which were produced by laser photolysis of hydrogen peroxide. It was found that the resulting methionine (Met) labeling pattern was consistent with the known BR structure. This finding demonstrates that laser-induced oxidative Met labeling can provide structural information on membrane proteins. In subsequent experiments, the effects of different denaturing agents (heat, acid, and SDS) on the BR conformation were investigated. It was demonstrated that each of these non-native conditions results in unique structural features that give rise to characteristic Met labeling patterns. These results highlight the ability of laser-induced oxidative labeling to detect conformational changes of membrane proteins.

Obtaining better insights into the structural properties of SDS-denatured BR is particularly important because this form of protein is widely used as starting point for folding studies. Combining oxidative labeling with site-directed mutagenesis and fluorescence measurements, this work yielded a detailed structural model of SDS-denatured BR. Subsequently, pulsed oxidative labeling coupled with rapid mixing and MS was used to characterize short-lived intermediates that become populated during BR refolding. The combination of pulsed oxidative labeling and stopped-flow spectroscopy provided key structural insights into the kinetic mechanism by which the SDS-denatured protein inserts and folds into the lipid bilayer.

Complementary to oxidative labeling, hydrogen/deuterium exchange (HDX) MS was employed to examine the structure and dynamics of BR under various physiochemical conditions. Structural features of different detergent/lipid-bound BR samples were characterized by their HDX kinetics. Comparative HDX experiments of BR were carried out in the dark (resting state) and under illumination where the induced retinal isomerization mediates proton transport (functioning state). Isotope exchange was found to be much faster during light exposure than in the dark. This observation reveals that structural dynamics of the protein scaffold are "accelerated" by motions of the retinal, reflecting a direct coupling between protein dynamics and function.