We study ATP synthase, a fundamental biological rotary motor that uses the proton motive force generated from metabolism to produce ATP, the universal chemical energy currency of life. ATP synthase is a multi-protein complex found in all forms of life and consisting of two coupled rotary motors. Our overall interest in this complex is the mechanism by which the electrochemical potential across the membrane is converted into rotary motion in the membrane-embedded Fo motor. Understanding the structural and biochemical basis for this process sheds light on a foundational life process, facilitates the design of antibiotics targeting the ATP synthase of pathogenic bacteria, and provides context for mutations of significance to human health.
Biochemical Assessment of Mutations at the Rotor-Stator Interface
Although the basic mechanism of proton transport is known, there are clusters of amino acids on the stator (subunit a) and the subunit c ring that have not been functionally characterized. Students use site-directed mutagenesis and chemical modification to probe the functional role(s) of these residues in various biochemical assays, including ATP-driven proton pumping and ATP synthesis assays.
Structural Dynamics of the Stator Subunit
The rotary motion of the rotor makes the Fo motor dynamic on a large scale, but it is unclear if smaller scale conformational changes in the stator subunit are involved in proton translocation. Students are purifying ATP synthases, reconstituting them into liposomes or nanodiscs, and using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to probe structural dynamics at various locations in the stator subunit.
Development of Antibiotic Compounds Targeting the Fo Motor
Microbial bioenergetics is an emerging target for antibiotic therapies, and several clinical and pre-clinical compounds specifically inhibit ATP synthase (e.g., bedaquiline for treatment of tuberculosis). Students are synthesizing and testing derivatives of quinolines to target several pathogenic bacteria, including Pseudomonas aeruginosa. (collaboration with Amanda Wolfe, UNCA Chemistry)
Characterization of Mutations Causing Neuromuscular Disorders in Humans
Mutations in human ATP synthase can impair mitochondrial function resulting in profound neurological effects. Since it is more difficult to conduct biochemical studies of human ATP synthase in vitro, and the basic structure and mechanism is the same in bacteria, we study equivalent bacterial mutations to observe their effects on the functions of the ATP synthase complex to shed light on the effects of the human mutations. Specifically, we are interested in rare mutations that lead to muscular dystonia and spastic paraplegia.