Analysis of the Transfer RNA Binding Effects on Cytochrome c and the Interactions of Tafazzin with Lipid Membrane by Hydrogen/Deuterium Exchange Mass Spectrometry
| Autor: | LO, YI-TING, 羅伊婷 |
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| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (ESI-MS) has been widely used to study the mechanisms of protein dynamics, domain structure, protein-ligand interactions and protein conformational changes.The redox protein cytochrome c plays an important role as an electron carrier in mitochondrial respiration. Release of cytochrome c from mitochondria triggers an intrinsic apoptosis pathway. The cytosolic transfer RNAs(tRNA) bind to cytochrome c preventing cytochrome c interaction with Apaf-1, blocking the formation of the apoptosome complex. To understand the interactions between tRNA and cytochrome c, we use HDXMS to analyze tRNA interaction with cytochrome c.Trnaphe and natural total tRNA are isolated from brewer’s yeast.tRNA binds to cyt.c to decrease the deuteration level, indicating the tRNA has caused cytochrome c to form a more compact conformation. To clarify the cause of binding, we use unstructured single strand oligonucleotidesto form complexes with cyt c in the HDXMS experiment. We compared with 12-mer dA and dT, although adenine and thymine are not important factors for the interactions, but cytochrome c in two regions 1-10 and 65-82 still showed to decreases upon unstructured dA or dT binding. Insummary, we conclude that N-terminal 20-32 is the selective region interacts with tRNA and N-terminus 1-10 interacts with oligonucleotides electrostatically. Tafazzin is an acyl transferase responsible for the remodeling of mitochondrial cardiolipin (CL). Natural mutation caused aberrant tafazzin in the Barth syndrome patients lost the remodeling function, leading to the abnormity of CL and monolyso-CL (MLCL) content. The activities of the tafazzin enzyme are tightly related to its interactions with lipid membrane. The 20% CL/ 80% PC phospholipid vesicle was prepared to simulate the mitochondrial membrane. We observed the interactions of the purified tafazzin with the CL vesicles by hydrogen/deuterium exchange mass spectrometry (HDXMS). Tafazzin protein contains a His69/Asp74 active site. The active sites and the surrounding loops show significant decreases of deuteration, which are potentially the membrane insertion regions. Based on the mechanism of the model of phospholipases, we suggest the surrounding loops positioned actives sites for the extraction of phospholipids upon lipid vesicle interaction. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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