All ETDs from UAB

Advisory Committee Chair

Bingdong Sha

Advisory Committee Members

Chenbei Chang

James Collawn

Champion Deivanayagam

Narayana Sthanam

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine


Tim44 is a peripheral membrane protein and a component of the TIM23 translocon on the matrix side. It is well established that Tim44 tethers the presequence associated motor (PAM) complex to the Inner Mitochondrial Membrane (IMM), through its C-Terminal Domain (CTD). This study focuses on understanding the high resolution structure of Tim44 CTD and the molecular basis for its membrane anchoring mechanism. The crystal structure of Tim44 CTD revealed that it exists as a single domain. The N-terminal amphipathic helices A1 and A2 protrude away from the main body of Tim44 CTD. These two flexible helices have been tested for their putative role in membrane anchoring by functional analyses. Biochemical studies were conducted using mutant forms of Tim44 CTD. Deletion and site directed mutants of Tim44 in the A1 and A2 helical region were used to study if the membrane binding ability is hindered. The deletion mutants included two constructs in which either a part of the A1-A2 helical region or the entire A1-A2 helical region was removed. The results showed that the A1 and A2 helices are required for Tim44 CTD to bind membranes. The point mutants included three constructs in which three conserved residues within A1 and A2 helices were mutated. Hydrophobic point mutations compromised the membrane binding ability. These investigations along with other structural data suggested that the A1-A2 helical region undergoes a conformational change along a hinge during ii membrane binding. This hinge is present in the loop that follows the A2 helix and is composed of two conserved glycine residues. This conformational change exposes the hydrophobic residues of the A1 and A2 helices to the IMM. In a soluble form these helices conceals the hydrophobic residues towards the protein core but during membrane binding, these residues seem to get exposed and interact with the membrane. We propose a mechanism by which the A1 and A2 helices turn along the glycine hinge either to conceal or to expose their hydrophobic side-chains based on the nature of the surrounding environment. The studies enumerated in this dissertation reveal the structure of Tim44 CTD at an atomic resolution. The membrane anchoring region of Tim44 CTD has been revealed using functional studies. Utilizing the structural and the functional data, a plausible mechanism for Tim44 CTD membrane binding has been proposed and tested.