Understanding Multilevel Protein Communication of the Mitochondrial Carrier Family and the TIM23 Complex

Mitochondria are dynamic organelles that require an intricate network of protein communication that traverse multiple compartments and membrane bilayers but membrane proteins are notoriously difficult to study with traditional structure determination methods. This thesis investigates intra- and inter- membrane protein communication including residue-residue communications within a protein, between protein domains, as well as inter-protein communication between proteins of a complex and of the electron transport chain (ETC). The first part of this thesis utilizes coevolution analysis to determine novel insights into the structure and structural dynamics of the mitochondrial carrier family (MCF) and Tim23. The ADP/ATP Carrier (AAC) is the most abundant and widely studied transporter of the MCF and imports ADP into the mitochondrial matrix and exports ATP to the cytosol by an alternating access mechanism resulting in two alternate conformational states: one with the channel interior exposed to the cytosol and one exposed to the matrix. Coevolution analysis of the AAC identifies novel residue interactions integral to the transition and stabilization of the AAC. Tim23 is the core channel-forming protein of the translocase of the inner membrane (TIM23) complex that is responsible for the translocation and integration of approximately 70% of mitochondrial proteins. Tim23 is composed of a soluble N-terminal domain in the intermembrane space and a membrane bound channel domain. Although key structural regions for complex assembly and protein translocation have been identified there is no high-resolution structure of Tim23. The coevolution analysis presented here predicts the first model of the Tim23 channel domain and predicts residue interactions that suggest novel structural dynamics. The second part of this thesis investigates the inter-protein communication between subunits of the TIM23 complex. Assays of native protein assemblies, demonstrate how the lipid cardiolipin, supports proper TIM23 subunit association. The final part of this thesis focuses on understanding the effect of NAPQI, an acetaminophen metabolite, on the electron transport activity of complex II. A reductionist approach, in which complex II was reconstituted into model membrane systems, was necessary to determine the concentration dependent effect of NAPQI on complex II activity