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Dissertation
The folded, partially folded, misfolded, and unfolded conformations of cytochrome C probed by optical spectroscopy
Doctor of Philosophy (Ph.D.), Drexel University
Sep 2013
DOI:
https://doi.org/10.17918/etd-4320
Abstract
Cytochrome c has been a cornerstone of biophysical research since it began. Because it is so well characterized, this protein remains an important molecule for ground-truthing new paradigms in protein biophysics. It was one of the first proteins for which a crystal structure was resolved; since then it has served continuously a traditional model system for the fundamentals of electron transfer between proteins and protein folding studies. The work presented here is a spectroscopic study of the folded, partially folded and misfolded states of oxidized cytochrome c, primarily via circular dichroism, absorption and resonance Raman spectroscopies. Here I report the first self-consistent study of the thermal transitions of all protonation states of ferricytochrome c at low anion concentration. UV circular dichroism data indicate only modest unfolding of the proteins helical structure at extreme pH and temperature. Thermally induced conformational changes are assignable to the unfolding of the lowest ranking foldons. Thermodynamic analysis showed a statistically significant enthalpy-entropy compensation, pointing to common physical processes for the unfolding of all protonation states in a similar way, this result suggests that similar physical processes cause all the observed protonation states. Additionally, I report the discovery of a novel (meta)stable, partially unfolded state of cytochrome c, obtained upon incubation under alkaline oxidizing conditions for an extended timed period. This frustrated, misfolded state is reversible; it undergoes an extremely slow transition back to the fully folded state after reintroduction to folding conditions for an extended period of time. Spectroscopic studies of the novel structure suggest a pentacoordinated quantum mixed spin state of the heme, previously found only in ferricytochrome c and in class-3 peroxidases. Cytochrome c has been shown to acquire peroxidase activity on the surface of liposomes and on the inner membrane of mitochondria, for which a quantum mixed state of the heme iron may be a prerequisite.
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Details
- Title
- The folded, partially folded, misfolded, and unfolded conformations of cytochrome C probed by optical spectroscopy
- Creators
- Jonathan Barrot Soffer - DU
- Contributors
- Reinhard Schweitzer-Stenner (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Arts and Sciences; Chemistry; Drexel University
- Other Identifier
- 4320; 991014632053004721