Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Chemistry

Language

English (en)

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Michael Gross

Abstract

Fast photochemical oxidation of proteins: FPOP) has shown great promise in the elucidation of the regions of a protein's structure that are changed upon interaction with other macromolecules, ligands, or by folding. The advantage of this protein footprinting method is that it utilizes the reactivity of hydroxyl radicals to stably modify solvent accessible residues non-specifically in a microsecond. The extent of *OH labeling at sites assays their solvent accessibility. We have corroborated the predicted profoundly short timescale of labeling empirically, by FPOP-labeling three oxidation-sensitive proteins and examining their global FPOP product outcomes. The novel test developed to validate conformational invariance during labeling can be applied generally to any footprinting methodology where perturbation to protein structure by the footprint labeling is suspected. The stable modifications can be detected and quantified by the same proteolysis, chromatography, and mass spectrometry techniques employed in proteomics studies; however, proteomics software does not automatically report the residue-resolved full-sequence-coverage footprint information found in proteomics-like FPOP data. Here we report the development of software tools to facilitate a comprehensive and efficient analysis of FPOP data, and demonstrate their use in a study of barstar in its unfolded and native states. We next show that SO4-* can serve as an alternative non-specific labeling agent that can be generated by the FPOP apparatus on the same fast timescale as *OH. This demonstrates the tunable nature of FPOP. We have used FPOP to characterize the oligomeric structures of three human apolipoprotein E: ApoE) isoforms and a monomeric mutant in their lipid-free states. Only one isoform of ApoE is strongly associated with Alzheimer's disease; unfortunately, the structural reason for this association is not known, in part because no high resolution structure exists of any isoform. We find that the three common isoforms of ApoE are very similar in their solvent accessible footprint, that their oligomeric interactions involve several regions in the C-terminal domain, and that the N-terminal domain of each resembles the monomeric mutant's N-terminal domain, the truncated form of which has been characterized as a four-helix bundle. Finally, we find by FPOP that ApoE interacts with beta-amyloid peptide 1-42 at a specific site in its N-terminal domain.

DOI

https://doi.org/10.7936/K72R3PQ8

Comments

Permanent URL: http://dx.doi.org/10.7936/K72R3PQ8

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