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High quality purification and preliminary Cryo-EM characterization of Pyruvate Dehydrogenase Complex from Bovine Mitochondria

High quality purification and preliminary Cryo-EM characterization of Pyruvate Dehydrogenase Complex from Bovine Mitochondria
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Issue Date
2021. 2
The pyruvate produced by glycolysis is oxidized to CO2 and H2O through the tricarboxylic acid (TCA) cycle and the respiratory chain. The pyruvate goes through the pyruvate dehydrogenase complex (PDC) composed of three enzymes to enter the TCA cycle. The PDC serves as a link between glycolysis and the tricarboxylic acid cycle. It also serves as a key control tower for energy and metabolic homeostasis. Because this role, the PDC is a potential therapeutic target for various diseases. Previously, the mammalian PDC structure was not properly recognizable due to its low resolution at 50 Å resolution. The purpose of our research was to purify and study the structure of an intact mammalian PDC at a better resolution than before. We extracted mitochondria from bovine heart and purified the PDC from that mitochondria. However, there was a problem with purity because the PDC and other proteins were not separated by previous purification methods. In order to solve this problem, we purified the PDC through glycerol gradient. However, there was no way to confirm the purity of the extracted PDC. Regular blue negative stain (BN-PAGE) was not appropriate for PDC observation due to its 9 mega-Dalton size locating the protein band between the separating gel and stacking gel. We improved the BN-PAGE to confirm the purity of the PDC. Afterwards, the high purity of the sample was confirmed through negative stain electron microscopy (negative stain-EM). Various concentrations were tested out through negative staining-EM to pick the optimal concentration of PDC fit for cryo-EM. As a result of cryo-EM, we obtained a structure of PDC from bovine that is better than the previous PDC structure. Interestingly, the previous PDC structure and our PDC structure were different. The reason is assumed to be due to structual change from inactivation by phosphorylation of the PDC, because in our experiment, the PDP was removed in the purification step. To support this, previous studies have shown that phosphorylation of pyruvate dehydrogenase can cause a structural switch.
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GRADUATE SCHOOL[S](대학원) > BIOENGINEERING(생명공학과) > Theses (Master)
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