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Tuesday, April 24 • 2:20pm - 2:40pm
Structural Dynamics In The FO Motor Of ATP Synthase Revealed By Site-Directed Spin-Labeling And CW-EPR

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F1FO ATP synthase is present in all life and is responsible for the production of almost all adenosine triphosphate (ATP), the ubiquitous energy currency synthesized during cellular metabolism. The FO motor converts electrochemical potential into mechanical rotation, which drives conformational changes in the F1 facilitating the synthesis of ATP. The mechanism of rotation of the FO rotor (subunit c) ring is unclear, but there is some evidence that the stator (subunit a) is conformationally dynamic. This study looked for further evidence of a ratcheting mechanism of rotation, which would require the α- helices of subunit a that lie on the a-c interface to move during rotation. Site directed mutagenesis was used to introduce cysteine into several positions on subunit a. The mutant ATP synthase was purified, chemically modified with MTSSL, a Cys-reactive spin-label, and observed using electron paramagnetic resonance (EPR) spectroscopy. The EPR spectrum is sensitive to the label environment and reports on the mobility of the residue to which the spin label is attached. While pH-dependent differences in mobility were apparent at aL195 and aV86/I161, the multi-component spectra indicated the presence of unreacted and/or off-target spin label preventing conclusive analysis of the data. Optimizing purification methods to remove the contaminating components may improve the quality of the data.


Tuesday April 24, 2018 2:20pm - 2:40pm PDT
123 Zeis Hall