Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Dr. Lars Konermann


Since the emergence of electrospray ionization (ESI) mass spectrometry (MS) as a tool for protein structural studies, this area has experienced tremendous growth. ESI-MS is highly sensitive, and it allows the analysis of biological systems ranging in size from a few atoms to large multi-protein complexes. This work aims to solve questions in protein structural biology by using ESI-MS in conjunction with other techniques.

We initially apply ESI-MS for studying the monomeric protein cytochrome c (Chapter 2). The physical reasons underlying the irreversible thermal denaturation of this protein remain controversial. By utilizing deconvoluted charge state distributions, oxidative modifications were found to be the major reason underlying the observed behavior. The positions of individual oxidation sites were identified by LC-MS/MS-based tryptic peptide mapping.

Chapter 3 and 4 focus on noncovalent protein complexes. ESI allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these assemblies by MS. It remains somewhat unclear, however, in how far this approach is suitable for measuring binding affinities. We demonstrate that the settings used for rf-only quadrupoles in the ion path are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful Kd measurements. Overall, our data support the viability of the direct ESI-MS approach for determining binding affinities of protein–protein complexes in solution.

Having established suitable conditions for the analysis of noncovalent protein complexes, ESI-MS is applied for monitoring the folding and assembly of hemoglobin (Hb). The native structure of this protein comprises four heme-bound subunits. Hb represents an important model system for exploring coupled folding/binding reactions, an area that remains difficult to tackle experimentally. We demonstrate that efficient Hb refolding depends on the heme ligation status. Only under properly optimized conditions is it possible to return denatured Hb to its tetrameric native state with high yield. ESI-MS allows the observation of on-pathway and off-pathway intermediates that become populated during this highly complex self-assembly process. In summary, this work demonstrates that ESI-MS is a highly versatile tool for addressing questions at the interface of chemistry and structural biology.

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