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Exploring Respiratory Chain Supercomplexes with Non-Canonical Amino Acids
Location: East Ballroom
Mentor: Dr. Flavia Fontanesi
The electron transport chain (ETC) is a group of protein complexes located on the inner mitochondrial membrane that produce a proton gradient that supports the synthesis of ATP, the primary source of cellular energy. In various organisms, complexes I–IV have been observed to form “supercomplexes” (SCs), consisting of the association in varying stoichiometries of the individual complexes I-IV. Studies on SC function and relevance have been limited by the transient and easily broken interactions between complexes in a SC, as only intermolecular forces hold the individual complexes in a SC together. This project seeks to substitute existing amino acid residues with non-canonical amino acids (ncAAs) that can form covalent linkages between the complexes to “lock” the SC in place, using the organism Saccharomyces cerevisiae as a model system. Multiple residues in the Cor1 subunit complex III – a primary contact point in SCs with the Cox5a subunit of complex IV – were mutated to amber codons which allow for the insertion of the ncAA. These mutations were screened in various stages to ensure the system was working as desired. Spot tests and western blots were used to screen for strains that properly synthesized the cor1 subunit when supplemented with the ncAA. Strains that synthesized the Cor1 subunit successfully were then subject to respirometry to assess the functionality of the Cor1 subunit before moving on to the cross-linking stage of the project.
