Presentation Type

Poster

Faculty Mentor’s Full Name

Bruce Bowler

Faculty Mentor’s Department

Biochemistry

Abstract

Cytochrome c (Cytc), a membrane associated redox active protein, functions as an electron shuttle in the electron transport chain. Additionally, it participates in the intrinsic pathway for apoptosis, exhibiting peroxidase activity when interacting with the mitochondrial membrane lipid, cardiolipin. The electrostatic binding site of Cytc, site A, has historically been attributed to positively-charged lysines, in particular, the Lys 72/73 and Lys 86/87 subclusters. These charge subclusters are thought to be the predominant site of interaction between cardiolipin and Cytc at pH 8 as they create zones of positive charge on the protein surface that interact with the negative charge of the cardiolipin headgroup. Recent research indicated that single, pairwise, and quadruple mutations of these Lys residues to the uncharged amino acid Ala in yeast iso-1-Cytc caused little change in binding to cardiolipin, indicating that site A may be more extensive than previously believed. In order to determine the importance of each subcluster to the overall binding affinity of site A in a higher eukaryote with a complete apoptotic pathway, double mutants of Human Cytc, Lys8687Ala and Lys7273Ala were created using PCR-based mutagenesis, characterized through dideoxy sequencing and then expressed in Escherichia coli. The binding constant, Kd, will be measured using fluorescence correlation spectroscopy (FCS) for mutants containing zinc substituted heme in the presence of nanodiscs containing 100% cardiolipin. By comparing these measurements with previous results for wild type Human Cytc, the importance of each of the subclusters to overall binding affinity can be determined by comparing the differences in Kd. Based on previous results with yeast iso-1-Cytc, it is expected that introduction of pairwise mutations will have little effect on Kd, contradicting the notion of a primary charge subcluster.

Category

Physical Sciences

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The Importance of Charge Subclusters on Overall Electrostatics of Human Cytochrome c Site-A Binding to Cardiolipin Nanodiscs

Cytochrome c (Cytc), a membrane associated redox active protein, functions as an electron shuttle in the electron transport chain. Additionally, it participates in the intrinsic pathway for apoptosis, exhibiting peroxidase activity when interacting with the mitochondrial membrane lipid, cardiolipin. The electrostatic binding site of Cytc, site A, has historically been attributed to positively-charged lysines, in particular, the Lys 72/73 and Lys 86/87 subclusters. These charge subclusters are thought to be the predominant site of interaction between cardiolipin and Cytc at pH 8 as they create zones of positive charge on the protein surface that interact with the negative charge of the cardiolipin headgroup. Recent research indicated that single, pairwise, and quadruple mutations of these Lys residues to the uncharged amino acid Ala in yeast iso-1-Cytc caused little change in binding to cardiolipin, indicating that site A may be more extensive than previously believed. In order to determine the importance of each subcluster to the overall binding affinity of site A in a higher eukaryote with a complete apoptotic pathway, double mutants of Human Cytc, Lys8687Ala and Lys7273Ala were created using PCR-based mutagenesis, characterized through dideoxy sequencing and then expressed in Escherichia coli. The binding constant, Kd, will be measured using fluorescence correlation spectroscopy (FCS) for mutants containing zinc substituted heme in the presence of nanodiscs containing 100% cardiolipin. By comparing these measurements with previous results for wild type Human Cytc, the importance of each of the subclusters to overall binding affinity can be determined by comparing the differences in Kd. Based on previous results with yeast iso-1-Cytc, it is expected that introduction of pairwise mutations will have little effect on Kd, contradicting the notion of a primary charge subcluster.