Structure function analysis of the conserved WWD domain in the System I bacterial holocytochrome C synthase, CcmF
Date
2022
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Publisher
University of Delaware
Abstract
Cytochromes c are highly conserved proteins found in nearly all organisms including bacteria, plants and humans that function in electron transport chains for critical cellular functions 1. Each cytochrome c contains at least one molecule of covalently attached heme which is required for protein folding, stability and function 1–3. The covalent attachment of heme to apocytochrome c is referred to as cytochrome c biogenesis. There are three known pathways that accomplish heme attachment within the inner membrane space of the mitochondria in eukaryotes (System III) or the periplasm of prokaryotes (System II and I) 1. All Systems function to covalently attach heme to apocytochrome c yet use different mechanisms. This thesis will focus on the integral membrane protein, and holocytochrome c synthase, CcmFH, of System I which attaches heme to apocytochrome c in the final step of the pathway 4. CcmF has key features that are required for function including four conserved histidines, a stable b-heme located in the transmembrane domain and a highly conserved WWD domain that has been shown to directly interact with heme in other proteins 5,6. Therefore, I hypothesize that the WWD domain in CcmF is required for holocytochrome c synthase activity. ☐ To test this hypothesis, alanine scanning was performed on the WWD domain in CcmF to determine which amino acids are necessary for cytochrome c biogenesis. Further biochemical analyses of each alanine mutant determined the heme environment in each variant and their protein interaction partners. These studies have shown that two alanine mutants in the WWD domain of CcmF displayed non-functional phenotypes. The inability of these two variants to perform the holocytochrome c synthase function was not due to a defect in heme binding nor to the disruption of known protein-protein interactions. Overall, this study demonstrates that the WWD domain is necessary for synthase function in CcmF.
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Keywords
Kinetic modeling, Pervades parameters, Feedstocks, Bayesian Inference Toolbox, Learning framework