Optimization of a Bacterial Two-Hybrid Assay to Determine the Optimal Temperature and E. Coli Strain for Cytochrome c Biogenesis System I protein:protein interactions

• Narra Swanson, Biological Sciences, University of Delaware

• Nikita Varde, Biological Sciences, University of Delaware
• Molly Sutherland, Biological Sciences, University of Delaware

Authors: Narra E. Swanson, Nikita Varde, Molly C. Sutherland

Cytochromes c are highly conserved proteins found in eukaryotes and prokaryotes that play important roles in respiration, electron transport, and photosynthesis. Cytochrome c requires covalent attachment of heme to a conserved CXXCH motif in apocytochrome c, a process known as cytochrome c biogenesis. Heme attachment is required for the proper folding and function of cytochrome c and can occur via three pathways: System I (Prokaryotes), II (Prokaryotes), or III (Eukaryotes). My summer project focuses on the System I pathway. System I consists of eight cytochrome c maturation proteins, CcmABCDEFGH, which are proposed to function in two steps: First, heme is transported across the membrane by the ABC transporter release complex, CcmABCD, for its attachment to CcmE. Once heme is attached to CcmE, the holoCcmE is released from the complex by ATP hydrolysis. Second, holoCcmE transports the heme to CcmFH for attachment to apocytochrome c. For heme attachment to occur, the cysteine thiols of the CXXCH motif must be in the reduced state and this reduction is catalyzed by the thioredoxin CcmG. While the functions of individual System I proteins have been examined in literature, the interactions between them still need to be studied. Two models for System I function have been proposed: The 2-Step Model and the Supercomplex model. According to the 2-Step Model, System I proteins form two protein subcomplexes (CcmABCD and CcmFGH) with CcmE interacting with both the complexes. In contrast, the Supercomplex Model proposes System I is a single multi-subunit supercomplex. To test these two models, a Bacterial Two-Hybrid (BACTH) Assay will be used for detecting and characterizing the protein-protein interactions in vivo. The BACTH assay uses blue-white colony screening to qualitatively and quantitatively assay β-galactosidase activity via reconstitution of T25 and T18 adenylate cyclase protein fragments. System I proteins CcmA, CcmE and CcmF, will be fused to T25 and or T18 to determine if these proteins interact. Using the positive and negative controls I determined the optimal temperature and dilution series for blue-white screening. Long term, these studies will elucidate the model of System I cytochrome c biogenesis pathway by the determination of protein-protein interactions.