Date of Completion


Embargo Period



mitochondria, protein, cardiolipin, membrane, import

Major Advisor

Nathan Alder

Associate Advisor

Andrei Alexandrescu

Associate Advisor

Eric May

Associate Advisor

Simon White

Associate Advisor

Andrew Wiemer

Field of Study

Molecular and Cell Biology


Doctor of Philosophy

Open Access

Open Access


Mitochondria acquire the vast majority of their proteins from the cell cytosol in a manner that requires the coordinated function of numerous translocation machineries embedded within their two membrane systems. One of the most multifaceted of these machineries is the Translocase of the Inner Membrane 23 (TIM23) complex, which mediates the import of both inner membrane- and matrix-targeted proteins. The central subunit of this complex is Tim23, a voltage-gated channel-forming protein that constitutes part of the protein-conducting channel residing in the inner membrane. Tim23 has bipartite domain organization with a C-terminal integral membrane domain and an N-terminal intrinsically disordered domain that resides in the intermembrane space, termed Tim23IMS. Work from our group and others have shown that cardiolipin, a dianionic phospholipid unique to the mitochondrion, is required for the activity and subunit interactions of the TIM23 complex, as well as for specific membrane interactions of Tim23IMS. Although the association between Tim23IMS and cardiolipin has been demonstrated, there is currently very little information regarding the molecular details of this protein-lipid binding interaction. This thesis focuses on elucidating the mechanism by which Tim23IMS binds to cardiolipin-containing membranes. We first discuss the theories and principles of spectroscopic techniques and data analysis strategies that can be utilized to effectively quantify transient binding interactions between intrinsically disordered proteins and model membrane systems. We next show how implementation of these techniques has allowed us to quantify Tim23IMS-membrane binding and critically assess the physicochemical driving forces behind this interaction. Using site-directed mutagenetic and computational approaches, we have identified highly-conserved amino acid residues of Tim23IMS that have proven critical in allowing this domain to bind to model membranes. Second, we show that cardiolipin facilitates the binding of Tim23IMS to mitochondrial membranes due to its unique structural properties. Finally, this work addresses the effects of Tim23IMS interactions on the physical properties of cardiolipin-containing bilayers themselves. Overall, our results have shifted the paradigm that cardiolipin and protein binding is primarily driven by electrostatic forces and have opened the door for future studies probing the structural properties of known cardiolipin-interacting proteins and mitochondrial membranes.