Researcher(s)
- Juliette MacNicoll, Biological Sciences, University of Delaware
- Evelyn McQuaid, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Ray Tsao, Biological Sciences, University of Delaware
- Nikita Varde, Biological Sciences, University of Delaware
- Molly Sutherland, Biological Sciences, University of Delaware
Abstract
Cytochromes c are proteins found in nearly every organism from humans to bacteria that mediate critical functions such as cellular respiration. Cytochromes c require the covalent attachment of heme to a conserved CXXCH motif for proper folding and function. This heme attachment is called cytochrome c biogenesis and can occur via one of three protein pathways: System I, II and III. My project focuses on the prokaryotic System II pathway from Bacteroides thetaiotaomicron, which is composed of two proteins CcsB and CcsA. It is known that the active site is made up of a heme-handling WWD domain that positions heme for attachment to apocytochrome c. However, we hypothesize interactions outside of the active site mediates specificity of heme attachment. A recent computational study identified a ‘beta-cap’ region in the periplasmic domain of CcsBA and proposes it functions to block the active site when heme is absent. We hypothesize that this ‘beta-cap’ plays a crucial role in the CcsBA-cytochrome c interaction. In order to test this hypothesis, my project utilized QuikChange site directed mutagenesis to mutate the following residues to alanine (F136A, Y141A, N171A, F136A/Y141A, Y141A/N171A, F136A/Y141A/N171A), to create variants that will be used in future assays to assess their impact on cytochrome c biogenesis. These site directed variants were engineered in a codon-optimized B. theta CcsBA gene for heterologous expression in E. coli. Next, these variants will be used for in vivo cytochrome c biogenesis assays to determine if these mutations impact heme attachment. Ultimately, this region could be a novel antibacterial target as the prokaryotic System II pathway is essential in a number of pathogenic organisms and has an active site that differs from that of human cytochrome c synthase.