Title

Electrostatic interactions of Cytochrome C and Cardiolipin: Quantitative analysis of structural changes of Cytochrome C by spectrometric techniques

Presentation Type

Poster

Abstract

Cytochrome c (Cytc) has a well-established role reacting with superoxide radicals to form molecular oxygen within the mitochondria. Cytc is bound to the inner mitochondrial membrane via the phospholipid cardiolipin. When Cytc dissociates from cardiolipin and crosses into the cytoplasm, it joins Apoptotic protease activating factor 1 (Apaf-1) to initiate programmed cell death. In the presence of reactive oxygen species (ROS), Cytc exhibits peroxidase activity which oxidizes cardiolipin and causes this dissociation. Additionally, Cytc can dissociate from cardiolipin in response to traumatic brain injury. We have worked to characterize the binding between Cytc and cardiolipin, information which may prove useful in developing a treatment for traumatic brain injury. In particular, the interaction between the anionic binding site (site A) and the phosphate head group of cardiolipin remains unclear. To study this interaction, we have mutated two amino acids postulated to be involved at this binding site. These amino acids, two lysine residues at positions 86 and 87, were replaced with alanine residues through point mutations introduced by PCR. Analysis of conformational changes of Cytc upon cardiolipin binding was performed by monitoring changes in fluorescence from Cytc’s tryptophan residue, an amino acid located near the active site. Samples were prepared utilizing UV-Vis spectroscopy and light scattering techniques to quantify experimental parameters. In conclusion, our analysis showed increased binding between Cytc and cardiolipin when the two lysine residues at 86 and 87 were replaced with alanines. This suggests that, contrary to initial speculation, that these two lysine residues help to destabilize the binding of Cytc to cardiolipin.

Category

Physical Sciences

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Electrostatic interactions of Cytochrome C and Cardiolipin: Quantitative analysis of structural changes of Cytochrome C by spectrometric techniques

Cytochrome c (Cytc) has a well-established role reacting with superoxide radicals to form molecular oxygen within the mitochondria. Cytc is bound to the inner mitochondrial membrane via the phospholipid cardiolipin. When Cytc dissociates from cardiolipin and crosses into the cytoplasm, it joins Apoptotic protease activating factor 1 (Apaf-1) to initiate programmed cell death. In the presence of reactive oxygen species (ROS), Cytc exhibits peroxidase activity which oxidizes cardiolipin and causes this dissociation. Additionally, Cytc can dissociate from cardiolipin in response to traumatic brain injury. We have worked to characterize the binding between Cytc and cardiolipin, information which may prove useful in developing a treatment for traumatic brain injury. In particular, the interaction between the anionic binding site (site A) and the phosphate head group of cardiolipin remains unclear. To study this interaction, we have mutated two amino acids postulated to be involved at this binding site. These amino acids, two lysine residues at positions 86 and 87, were replaced with alanine residues through point mutations introduced by PCR. Analysis of conformational changes of Cytc upon cardiolipin binding was performed by monitoring changes in fluorescence from Cytc’s tryptophan residue, an amino acid located near the active site. Samples were prepared utilizing UV-Vis spectroscopy and light scattering techniques to quantify experimental parameters. In conclusion, our analysis showed increased binding between Cytc and cardiolipin when the two lysine residues at 86 and 87 were replaced with alanines. This suggests that, contrary to initial speculation, that these two lysine residues help to destabilize the binding of Cytc to cardiolipin.