Graduation Year

2021

Graduation Month

May

Document Type

Thesis

Degree Name

Bachelor of Science

School or Department

Chemistry and Biochemistry

Major

Biochemistry – Health Professions

Faculty Mentor

Bruce E. Bowler

Faculty Mentor Department

Chemistry and Biochemistry, Department of

Keywords

Cytochrome c, peroxidase, apoptosis

Abstract

The mitochondria is an important organelle for a large assortment of metabolic processes. Cytochrome c (Cytc), a vital protein found in the mitochondria, is critical for life and death processes in eukaryotic cells. One of these functions includes shuttling electrons through the electron transport chain during cellular respiration. Cytc is also an essential signaling protein in the pathway of apoptosis, also known as programmed cell death. Yeast has similar components of this cell death pathway to higher eukaryotes such as humans, but does not contain all of the same optimized cascade processes. Additionally, the peroxidase activity, an early signal in the apoptotic pathway, is much lower in human Cytc when compared to wild type yeast iso-1- Cytc. This suggests the evolution of an optimized “off” state in the peroxidase activity of human Cytc. In the least stable substructures of Cytc, which mediate peroxidase activity, three amino acid sites co-evolve between human Cytc and iso-1-Cytc. In comparison to iso-1-Cytc, human Cytc contains the substitutions S40T, V57I and N63T. These should stabilize this hydrophobic interface in human Cytc by the addition of a methyl group in each substitution when compared to iso-1-Cytc. All possible single, double and triple substitution variants from Hu Cytc were introduced into iso-1-Cytc to test the hypothesis that they would increase the stability of iso-1- Cytc, causing the peroxidase activity of iso-1-Cytc to decrease. Bacterial protein expression of the single and double mutation variants in BL21 Escherichia coli cells, followed by protein purification were performed. Further experimentation on the individual mutant proteins, to test the hypothesis, included measurement of peroxidase activity, the alkaline transition, and protein stability. Overall we find only moderate effects on global and local stability by each of the variants introduced. While there are minor changes in the KM for each of the variants in comparison to WT, kcat shows more variation between WT and variants at increasing pH values. These findings suggest that these variants evolved to preserve rather than change function.

Honors College Research Project

Yes

GLI Capstone Project

no

Available for download on Sunday, May 01, 2022

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© Copyright 2021 Sidney L. Thompson