Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Lars Konermann

Abstract

A key challenge associated with protein folding studies is the characterization of short-lived intermediates that become populated en route to the native state. In this work, a covalent labeling method was developed that provides insights into the structures of these transient species. Hydroxyl radical (·OH) reacts with solvent-exposed side chains, whereas buried residues are protected. Mass spectrometry is used for monitoring the locations and the extent of labeling. Pulsed ·OH labeling of proteins at selected time points during folding results in high temporal and spatial resolution when compared to existing other labeling methods.

This novel technique was validated by studying the kinetic unfolding and refolding of holomyoglobin (hMb) and cytochrome c (cyt c), respectively. The noncovalent prosthetic heme group in hMb was shown to drastically affect the unfolding pathway. Cyt c refolding was found to fold in a stepwise manner. The population of a misfolded cyt c intermediate was also detected. Results in both cases were in accord with published data.

Many cellular proteins exist as oligomers. Pulsed ·OH labeling method was therefore extended to monitor the folding and assembly of a 22 kDa homodimeric protein, S100A11. Prior to this study very little information regarding the folding mechanism of this protein was available. ·OH labeling reveals that disruption of the native dimer is followed by the formation of non-native hydrophobic contacts within the denatured monomers. The folding/binding pathway was shown to progress through monomeric and dimeric intermediates.

In the final section of this study we applied ·OH labeling to a large monomeric protein that folds to a metastable state. The folding pathway of the 44 kDa protease inhibitor, α1-antitrypsin, was characterized and compared with complementary data from hydrogen/deuterium exchange studies. Our results show that the formation of early tertiary contacts and specific hydrogen bonds guide the protein towards its active, metastable structure. Structural correlation is also seen between a late kinetic species and a previously characterized equilibrium intermediate of a pathogenic mutant.

Overall, the results presented highlight the ability of the technique developed in this work to provide in-depth information about the mechanisms of protein folding.

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