The study of colorimetric pH probe and optical detection of heme attachment to cyt C by using genetically encoded indicators in living cells

Abstract

The cytochrome c (Cyt c) is a metalloprotein that has a heme as a cofactor. The heme has a reduced iron atom (Fe2+) on the center of its structure. The Fe2+ atom is important for formation of thioether bonds. The thioether bonds are formed between the thiol groups of Cys18 and Cys15 residues of apocytochrome c (apocyt c) that is unfolded forms of Cyt c and the vinyl groups of heme by catalyzing of holocytochrome c synthase (HCCS) or cytochrome c heme lyase (CCHL). Met81 coordination change also affects the folding of Cyt c. The Cyt c has two main function in human cells. One of them is to transfer the electron from Cyt bc1 to Cyt c oxidase in electron transfer system (ETS) which is an important step to reduce oxygen to water in respiration. Another function is to bind apoptotic protease activating factor-1 (Apaf-1) complex to activate caspases in intrinsic pathway of apoptosis.The Cyt c is encoded in CYCS gene which is located on 7p15.3 region of chromosome 7. The substitution mutation of G41S on CYCS gene in megakaryocytes results in an autosomal dominant genetic disorder called thrombocytopenia. In this case, the platelet cannot form their mature forms due to enter apoptosis in their premature forms and does not have a function. The CCHL is encoded on HCCS gene which is located on Xpter region of X chromosome and is an evolutionary conserved gene. The rearrangement of Xpter to Xp22 results in a neurodevelopment genetic disorder called as microphthalmia with linear skin defects syndrome (MLS). The MLS is lethal for males since they have only one X chromosome, whereas the females have two X chromosome and they show the disease in phenotype due to random X inactivation. The Cyt c is detected and studied by various biological methods. The Western blot, enzyme linked immunosorbent assay (ELISA) and flow cytometry are extensively used to understand the role of Cyt c in cells. These methods use specific antibodies to detect Cyt c. Circular dichroism (CD), the hydrogen/deuterium (H/D) exchange, the Fourier transform infrared (FTIR), gel filtration and the isothermal titration calorimetry (IATC) are spectroscopic techniques that were used to study the structural changes of Cyt c. Although all of these techniques are sensitive to detect Cyt c in vitro, they are not useful to study conformational changes Cyt c in vivo especially in living cells at spatial and temporal resolution.The genetically encoded fluorescence probes enable to visualize cellular functions in living cells and organisms in real time. The green fluorescence protein (GFP) is the first isolated protein from Aequorea victoria. The fluorescence protein gene is cloned into a vector and the desired protein gene is also cloned upstream or downstream of fluorescence protein gene. Then, the vector is transformed into the cell such as a bacteria or cell line and expressed in the cell recombinantly. Then, the fluorescence probe characterized by spectroscopic techniques and used to follow cellular functions in vivo in real time by light microscope. The fluorescence resonance energy transfer (FRET) system is a kind of genetically encoded fluorescence probe that uses two different fluorescence proteins. One of them is the acceptor that is placed to upstream of the desired protein. Another is the donor that is placed to downstream of the desired protein. There are actually different types of FRET such as ligand-dependent FRET probes and catalytic probes. For example, there is a sensor domain between acceptor (CFP) and donor (YFP) proteins. If a ligand binds to sensor domain, there is a conformational change occurs and results in the approaching of acceptor and donor with each other. In this case, the acceptor absorbs the signal from laser microscope and the donor quenches the signal from acceptor, emits a FRET signal to detector. In this study, we firstly characterized a FRET construct called Cyt c-Venus as a colorimetric and ratiometric pH probe and compared to the other control constructs which are Cyt c and Apocyt c-Venus. These constructs were designed by recombinant cloning methods in our previous study, but their pH sensitivity were not characterized in detail. We expressed these constructs in BL21 DE3 E.coli bacterial cells and treated with pH 4.5, 5.5, 6.5, 7.5 and 8 PBS buffers. We observed that there are changes in the colors of Cyt c-Venus and Apocyt c-Venus, but there is no change in the color of Cyt c under visible light, as the pH declines from 8 to 4.5. We observed fluorescences of Cyt c-Venus and Cyt c in pH 8 and 7.5, but the fluorescence of Cyt c-Venus and Apocyt c-Venus were diminished in pH 6.5, 5.5 and 4.5, whereas there is no fluorescence in Cyt c in all pH values since it does not have fluorescence protein under UV light. The absorbances of these proteins were measured by UV-Vis spectrophotometer. We observed that there were two signal peaks in Cyt c-Venus in pH 8 and 7.5. One of them is at 408 nm which belongs to Cyt c, another is at 515 nm which belongs to Venus. The signal peak of Venus in Cyt c-Venus started to decrease, as the pH reduced and it was disappeared in pH 4.5. On the other hand, we did not observe any change in the signal peak of Cyt c in all pH values. To get smooth peaks in absorbance graphs and prevent background signals due to cellular wastes, we lysed the cells and purified the proteins by ion-exchange chromatography from other cellular waste components. Then, we run the proteins in SDS-PAGE. We successfully purified Cyt c and Cyt c-Venus. We titrated the pure Cyt c-Venus protein by dropping mild HCI. We had observed that there was a color change in Cyt c-Venus compared to Cyt c and non-titrated Cyt c-Venus. Indeed, we had expected that the color of Cyt c-Venus turned from orange to red at acidic pH under visible light due to sensitivity of Venus to pH. Although it did not turn to red, orange color was reduced. We also observed a decline in its fluorescence compared to non-titrated Cyt c-Venus that had a brilliant fluorescence under UV light and compared to Cyt c that did not have any fluorescence. We measured the absorbance or excitation of Cyt c-Venus during titration and observed that there was a decline of signal peak of Venus at 515 nm and it finally disappeared at low pH values. There was no change at the signal peak of Cyt c at 408 nm. We also titrated pure Cyt c by mild HCI to use as a control and observed no change in the color, fluorescence and absorption signal of Cyt c.Secondly, we also investigated the heme binding properties and conformational changes of Cyt c by design a FRET probe. We placed a CFP on upstream of Cyt c as acceptor and a YFP on downstream of Cyt c as donor. Then, we expressed the construct in neuronal RPE cells.We calculated the netFRET values to exclude background signals and we normalized the netFRET values to normFRET values which are between 0 and 1. We also used C17V as positive control which has a 17 amino acid linker between Cerulean and Venus. We observed that there was a FRET signal in Cerulean-Cyt c-Venus construct in the presence of heme and heme lyase compared to C17V which has also FRET signal. We observed that there was an increase in FRET signal in Cerulean-Apocytc-Venus in the presence of only heme. On the other hand, there was no FRET signal in Cerulean-Apocytc-Venus construct in the absence of heme and heme lyase or in the presence of only heme lyase. As the concentration of heme increased, we observed that the FRET signal increased in Cerulean-Apocytc-Venus. The expression of heme lyase in the absence of heme did not change the FRET signal indicating that heme is important for binding of heme lyase and Cyt c. As a control, we separately expressed Cerulean and Venus and compared the FRET signal to C17V in the presence of heme. We observed that the heme did not bind to C17V.As a conclusion, we have characterized that the Cyt c-Venus is a sensitive protein construct to pH changes. It can be used to visualize pH changes in real time. We have used FRET method to determine the intermediate state of Cyt c in RPE cells. We have also found that the heme can bind to Cyt c non-covalently in the absence of heme lyase and causes conformational changes in Cyt c by using FRET method.