Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Chemistry

Language

English (en)

Date of Award

6-18-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Michael L Gross

Abstract

Two important biophysical characteristics of proteins, their interaction with ligands and their post-translational modifications, modulate various biological processes including signal transduction, chemical synthesis, and cell function. Protein-ligand interactions include the interactions with protein, peptide, DNA, and metal. Characterization of the physical properties of these interactions: binding interfaces, binding affinities, and the protein conformational changes due to the binding) is essential in understanding the mechanism of related diseases and, more importantly, in future drug design.

Mass spectrometry, with its own revolution and improvement, becomes a powerful tool in protein and peptide analysis. In this thesis, we applied two mass spectrometry-based strategies, proteomics and protein footprinting, to characterize these biophysical properties of three disease-related proteins, connexin 43: Cx43), troponin, and apolipoprotein E: ApoE), and Fenna-Matthews-Olson protein: FMO), which is the key factor in energy transfer of the photosynthetic system of green sulfur bacteria.

By the combination of standard proteomics workflow and two fragmentation methods, collision-induced dissociation: CID) and electron transfer dissociation: ETD), we successfully identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.

We further utilized hydrogen/deuterium exchange: H/DX), one mass spectrometry-based protein footprinting strategy, to examine the binding affinities of troponin C: TnC), a cardiac disease-related protein, with its four metal binding ligands: Ca2+), and their binding order. We then expanded this approach to elucidate the dynamics of TnC within the complex and its interactions with other subunits: TnT and TnI) at peptide-level resolution. This same approach was also applied to two protein-ligand complexes:: 1) the interaction of FMO and its binding partner, CsmA baseplate protein, in which the orientation of FMO between chlorosome and membrane can be confirmed, and: 2) the interaction of ApoE and Abeta 40, which are both key factors in Alzheimer's disease. Moreover, we improved the spatial resolution of H/DX to residue-level by conducting ETD fragmentation in the study of ApoE oligomerization. Our results reveal, for the first time, the amino acid residues involved in its self-oligomerization.

These six applications of mass spectrometry-based approaches show their potential in the characterization of different protein biophysical properties. The investigation of a more complex protein system can then be pursued.

DOI

https://doi.org/10.7936/K7FT8J23

Comments

Permanent URL: http://dx.doi.org/10.7936/K7FT8J23

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