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Poster Presentations
Day 3, June 24（Tue.）
Room P (Maesato East, Foyer, Ocean Wing)
- 3P-PM-33
Structural Characterization of Human Mitochondrial Single-Stranded DNA-Binding Protein and Helicase Twinkle Using Native Mass Spectrometry
(1NCKU CHEM, 2NCKU BIMB, 3NCKU BMS)
oTing-Yi Chiang1, Po-Jung Cien2, Chyuan-Chuan Wu3, Szu-Hsueh Lai1
Defects in maintaining mitochondrial DNA (mtDNA) can disrupt mitochondrial homeostasis and energy production in eukaryotic cells, leading to disease. Understanding the mechanisms of mtDNA replication is essential for elucidating the origins of mitochondrial disorders. The single-stranded DNA-binding protein (mtSSB) and the mtDNA helicase Twinkle play key roles in mtDNA replication and maintenance. Previous studies using Förster Resonance Energy Transfer (FRET) demonstrated mtSSB tetramer can bind to ssDNA in two different binding modes: (SSB)<SUB>30<SUB> and (SSB)<SUB>60<SUB>, corresponding to DNA sizes of 30 and 60 nucleotides, respectively. However, FRET relies on fluorescent labeling and cannot directly determine the protein's oligomeric state. In addition, the cryo-EM analysis of wild-type (WT) <I>Homo sapiens<I> (<I>Hs<I>) Twinkle structure remains challenging due to its high heterogeneity and instability, which complicate particle selection, classification, and the exclusion of unsuitable images. These challenges require extensive optimization to obtain reliable data. Native mass spectrometry (native MS) is complementary, preserving noncovalent interactions between the oligomeric protein complex and ssDNA. In this study, we employed native MS to characterize all intermediate states identified in the FRET study of mtSSB, primarily focusing on the interaction between the tetrameric mtSSB and ssDNA. Additionally, our preliminary findings provide direct evidence of WT <I>Hs<I>Twinkle oligomerization distribution.