Cytochrome Cproteininin redoks durumu ve maturasyonunu çalışmak için genetik olarak kodlanabilen görüntüleme metotlarının geliştirilmesi

Genetically encoded fluorescent reporters for biological signaling and advance microscopy techniques play a key role to elucidate the mechanism of changes in cells under oxidative stress, therefore provide quantitative methods to monitor signaling and measure response to external stimuli at higher spatiotemporal resolution. The use of these methods is highly preferable due to their low toxicity, high signal-to-noise ratio, allowing signal detection from various cell types and specificity for complex biological pathways. Redox mechanism is an essential pathway for living organisms in order to maintain and preserve biological homeostasis. It fulfills the completion of respiratory chain for energy production, cellular reduction, and regulation of free radical species and initiation of apoptosis. Oxidative damage and unbalanced redox propagation can facilitate deleterious situations like aging, neurodegenerative diseases, and hypoxia. Thus, a better understanding of redox mechanism included structures can reveal invaluable information on its interplay in intracellular signaling. Cytochrome c (Cyt c), a member of redox active heme proteins family, is localized in the inner membrane of mitochondria where a great number of intracellular redox reactions take place. Its main function is to transfer electrons across mitochondria's membrane in electron transport chain of adenosine triphosphate (ATP) production. Therefore, the key role of Cyt c in mitochondria's redox mechanism highlights it as a good candidate for advancing our knowledge on the redox biogenesis and lead the way to the diagnosis and treatment of many redox mechanism related diseases. Here, we demonstrated a method for monitoring and determining the redox state of Cyt c in microbial purified proteins and studied the maturation and interaction of Cyt c with Cyt c heme lyase in living cells with fluorescence resonance energy transfer (FRET). The oxidation state of Cyt c was determined through the photochromic FRET (pcFRET) method where the emission of the fluorescent reporter attached to Cyt c was modulated by the redox state driven absorption changes of Cyt c. The continuous changes of Cyt c oxidation state induced by various redox compounds were clearly demonstrated. Finally, we studied the heme lyase induced hemin insertion and folding of Cyt c in living cells and redox protein interactions by using a single-cell fluorescence signal tracking method. We also reported a novel method of frame-by-frame, fluorophore coefficient corrected FRET tracking for the characterization of the retrieved signal coming directly from living cells.