Mandal, Abhishek
(2017)
INVESTIGATIONS OF THE STRUCTURAL CHANGES IN PROAPOPTOTIC PEROXIDASE-ACTIVE CARDIOLIPIN-BOUND CYTOCHROME C AND LIQUID- GEL PHASE TRANSITIONS IN LIPOSOMES USING SOLID STATE NMR SPECTROSCOPY.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Mitochondrial cytochrome c (cyt-c) plays a key role in the activation of intrinsic apoptosis. Cyt-c gains a new function under apoptotic conditions; the peroxidation of mitochondrial lipid cardiolipin (CL) by cyt-c is a required step in the intrinsic apoptosis pathway. Understanding the mechanism of this alternate functionality in cyt-c has implications for treatment of neurological diseases like Huntington’s disease and in cancer. In order to gain insights into this mechanism, structural and dynamical information on the membrane bound protein is required. Solid-state nuclear magnetic resonance (ssNMR) provides an array of tools to study this system and extract necessary structural and dynamical information on the protein as well as the membranes. In this thesis, magic-angle-spinning (MAS) NMR and static ssNMR are used in conjunction with various other biophysical tools to gain insights into the mechanism of cyt-c’s peroxidase activity.
The effect of lipid peroxidation by cyt-c during apoptosis has also been implicated in modulating the structure, dynamics and behaviour of mitochondrial membranes, including facilitating pore formation in cyt-c-bound liposomes [1-3]. An understanding of lipid structure and phase behaviour has tremendous bearing on the study of lipids, membrane proteins and cryoprotection in general and cyt-c’s role in apoptosis in particular. MAS NMR is used here to investigate the link between the freezing point depression of water and the lowering of the lipid transition temperature.
One of the requirements to performing MAS NMR is a method of sample preparation that provides highly concentrated samples in tiny microliter sized MAS sample rotors. Additionally, for biological samples, as the ones under study here, it is very important to maintain hydration of the sample at all times in order to preserve function of the protein and the membranes, prevent damage to the sample, obtain better quality NMR spectra and most importantly, maintain biological relevance. Thus, the design and use of an ultracentrifuge based packing tool, that fulfills the requirements listed above, is discussed and illustrated here.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
4 January 2017 |
| Date Type: |
Publication |
| Defense Date: |
30 November 2016 |
| Approval Date: |
4 January 2017 |
| Submission Date: |
20 December 2016 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
138 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics and Structural Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
cytochrome c, cardiolipin, intrinsic apoptosis, solid state NMR, magic angle spinning NMR, peroxidase activity, protein structure, lipid phase change, MAS sample packing tool |
| Date Deposited: |
04 Jan 2017 13:51 |
| Last Modified: |
04 Jan 2018 06:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/30357 |
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