Effects of Oxidative Modifications on the Structure and Non- Canonical Functions of Cytochrome c Studied by Mass Spectrometry

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Effects of Oxidative Modifications on the Structure and Non-Canonical Functions of Cytochrome c Studied by Mass Spectrometry, Is A Well-Researched Topic, It Is To Be Used As A Guide Or Framework For Your Research

Abstract

The peroxidase activity of the mitochondrial protein cytochrome c (cyt c) plays a critical role in triggering programmed cell death, or apoptosis. However, the native structure of cyt c should render this activity impossible due to the lack of open iron coordination sites at its heme cofactor. Despite its key biological importance, the molecular mechanisms underlying this structure-function mismatch remain enigmatic. The work detailed in this dissertation fills this knowledge gap by using mass spectrometry (MS) to decipher the central role that protein oxidative modifications and their associated structural changes play in activating the peroxidase function of cyt c.

Chapter 2 uses a suite of MS-based experiments to identify and characterize oxidative modifications in cyt c caused by the oxidant and canonical peroxidase substrate, H2O2. In doing so, we unravel the critical role that these in situ structural changes play in triggering the peroxidase activity of the protein via alteration of the coordination environment. Serendipitously, we also discover that certain functionally important oxidative modifications, particularly on Lys, can elude detection when using conventional bottom-up MS approaches. However, by applying top-down MS we could successfully detect these modifications.

Chapter 3 re-examines a popular and purportedly well-characterized model system for peroxidase-activated cyt c: cyt c treated with chloramine-T. By combining top-down MS with sample fractionation techniques, we uncover that this model system is in fact comprised of a broad ensemble of structurally and functionally distinct species. These species can be differentiated by the extent of oxidation at key Lys residues, which previously went undetected.

Chapter 4 expands on the previous chapters by probing the causal factors underpinning the production of oxidative modification products at Lys and other residues. We discover that Lys oxidation is catalyzed by the endogenous heme cofactor, while other transformations (e.g. Met oxidation) proceed via direct interaction with the oxidant.

Chapter 5 utilizes oxidized cyt c as a model system to test the compatibility of protein stability measurements in the gas phase to their counterparts in solution. Unlike many other protein systems, we discover that oxidized cyt c shows opposing stability trends in solution and in the gas phase.

Table of Contents

Abstract ……………………………………………………………………………………………………………… ii
Summary for Lay Audience ……………………………………………………………………………….. iv
Co-Authorship Statement ………………………………………………………………………………….. vi
Acknowledgements ………………………………………………………………………………………….. viii
Table of Contents ………………………………………………………………………………………………. ix
List of Symbols and Abbreviations ……………………………………………………………………. xv
Chapter 1. Introduction ………………………………………………………………………………………. 1
1.1. Protein Structure and Function ………………………………………………………….. 1
1.2. Cytochrome c …………………………………………………………………………………….. 2
1.3. Protein Oxidation ………………………………………………………………………………. 6
1.4. Common Methods for Studying Protein Structure ………………………………. 7
1.4.1. Optical Spectroscopy …………………………………………………………………….. 7
1.4.2. High-Resolution Structural Techniques ……………………………………………. 8
1.5. Mass Spectrometry and Associated Techniques…………………………………… 9
1.5.1. Electrospray Ionization Mass Spectrometry (ESI-MS) …………………….. 10
1.5.2. Mass Spectrometers for Protein Analysis ……………………………………….. 11
1.5.3. Ion Mobility Spectrometry……………………………………………………………. 14
1.6. ESI-MS Experiments for Studying Protein Structure ………………………… 16

1.6.1. Primary Structure: Sequencing ……………………………………………………… 16
1.6.2. Higher Order Structure: Covalent Labeling Experiments …………………. 18
1.6.3. Higher Order Structure: Native Mass Spectrometry …………………………. 19
1.7. Scope of Thesis …………………………………………………………………………………. 20
1.8. References ……………………………………………………………………………………….. 22
Chapter 2. Elucidation of an H2O2-Induced, In Situ Activation Mechanism of the Peroxidase Activity of Cytochrome c ………………………………………………………. 34
2.1. Introduction …………………………………………………………………………………….. 34
2.2. Materials and Methods …………………………………………………………………….. 37
2.2.1. Materials. …………………………………………………………………………………… 37
2.2.2. Optical Spectroscopy. ………………………………………………………………….. 37
2.2.3. Protein Samples. …………………………………………………………………………. 37
2.2.4. NMR Spectroscopy. …………………………………………………………………….. 38
2.2.5. Mass Spectrometry. …………………………………………………………………….. 38
2.3. Results and Discussion ……………………………………………………………………… 39
2.3.1. Peroxidase Kinetics. …………………………………………………………………….. 39
2.3.2. Spectroscopic Evidence for H2O2-Induced Structural Changes. ………… 43
2.3.3. H2O2-Induced Modifications Probed by Mass Spectrometry. ……………. 46
2.3.4. Peptide Mapping of Oxidation Patterns. …………………………………………. 47
2.3.5. Chemical Deconvolution. …………………………………………………………….. 52

2.3.6. Top-down MS. ……………………………………………………………………………. 57
2.3.7. Peroxidase Activation Mechanism of Cyt c. …………………………………… 61
2.4. Conclusions ……………………………………………………………………………………… 63
2.5. References ……………………………………………………………………………………….. 64
Chapter 3. Lysine Carbonylation as a Previously Unrecognized Contributor to Peroxidase Activation of Cytochrome c in Oxidants Other than H2O2 ……… 70
3.1. Introduction …………………………………………………………………………………….. 70
3.2. Methods …………………………………………………………………………………………… 74
3.2.1. Materials. …………………………………………………………………………………… 74
3.2.2. Preparation of CT-cyt c. ……………………………………………………………….. 74
3.2.3. Optical Spectroscopy. ………………………………………………………………….. 74
3.2.4. Mass Spectrometry. …………………………………………………………………….. 75
3.3. Results and Discussion ……………………………………………………………………… 76
3.3.1. Chloramine-T-induced Met80 Oxidation. ………………………………………. 76
3.3.2. SCX Fractionation and Optical Characterization. ……………………………. 76
3.3.3. Peroxidase Activity of CT-cyt c Fractions. …………………………………….. 81
3.3.4. SCX Fractions Represent Specific Proteoforms. ……………………………… 82
3.3.5. Top-Down CID-IM-MS for Proteoform-Selective Analysis. …………….. 86
3.3.6. Mapping of LysCHO Sites. ………………………………………………………….. 91
3.3.7. LC-MS/MS Peptide Mapping Revisited. ………………………………………… 94

3.3.8. Mechanism of CT-induced Peroxidase Activation. ………………………….. 96
3.4. Conclusions ……………………………………………………………………………………. 100
3.5. References ……………………………………………………………………………………… 102
Chapter 4. Delineating Heme-Mediated versus Direct Protein Oxidation Pathways in Peroxidase-Activated Cytochrome c ……………………………………………………… 107
4.1. Introduction …………………………………………………………………………………… 107
4.2. Methods …………………………………………………………………………………………. 111
4.2.1. Materials. …………………………………………………………………………………. 111
4.2.2. Mass Spectrometry. …………………………………………………………………… 111
4.2.3. Heme Removal: Preparation of apoSS-cyt c. ………………………………….. 111
4.2.4. CT-induced Oxidation and GRT Labeling. …………………………………… 112
4.3. Results and Discussion ……………………………………………………………………. 115
4.3.1. Effects of CT-Induced Oxidation. ……………………………………………….. 115
4.3.2. Oxidation Site Mapping. …………………………………………………………….. 118
4.3.3. Confirming LysCHO Sites by GRT Labeling. ………………………………. 121
4.3.4. Confirming Heme Catalysis Using MP11. ……………………………………. 125
4.4. Conclusions ……………………………………………………………………………………. 128
4.5. References ……………………………………………………………………………………… 130

Chapter 5. Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase ……………………. 135
5.1. Introduction …………………………………………………………………………………… 135
5.2. Materials and Methods …………………………………………………………………… 138
5.2.1. Materials. …………………………………………………………………………………. 138
5.2.2. Protein Oxidation. ……………………………………………………………………… 139
5.2.3. Thermal Unfolding. …………………………………………………………………… 139
5.2.4. Mass Spectrometry. …………………………………………………………………… 140
5.3. Results and Discussion ……………………………………………………………………. 141
5.3.1. Chromatographic Separation of Proteoforms. ……………………………….. 141
5.3.2. Stability of CT-cyt c in Solution. …………………………………………………. 143
5.3.3. ESI Charge States of CT-cyt c. ……………………………………………………. 145
5.3.4. Native ESI Gas Phase Conformations of CT-cyt c. ………………………… 147
5.3.5. Gas Phase Stability of CT-cyt c. ………………………………………………….. 147
5.3.6. Proteoform-Resolved CIU Analysis. ……………………………………………. 151
5.4. Conclusions ……………………………………………………………………………………. 154
5.5. References ……………………………………………………………………………………… 156
Chapter 6. Summary and Future Work …………………………………………………………… 161
6.1. Summary ……………………………………………………………………………………….. 161
6.2. Future Work ………………………………………………………………………………….. 162

6.2.1. Peroxidase Activation In Vivo …………………………………………………….. 162
6.2.2. Cyt c / Cardiolipin Interactions ……………………………………………………. 163
6.2.3. Oxidation Mapping in Other Systems ………………………………………….. 163
6.3. References ……………………………………………………………………………………… 165
Appendix I – Permissions ………………………………………………………………………………… 167
Curriculum Vitae ……………………………………………………………………………………………. 170

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Additional information

Author

Victor Yin

No of Chapters

6

No of Pages

188

Reference

YES

Format

PDF

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