Abstract

As the primary producer of a cell’s energy currency, adenosine triphosphate (ATP), F1FO ATP synthase is essential for life on this planet. Using the H+ gradient across a membrane, the Fo complex generates torque on the F1 complex, which synthesizes ATP. However, aspects of Fo functionality, including the functional interactions between subunit a (stator) and the c10 ring (rotor) remain unresolved. Through site-directed mutagenesis and chemical modifications we found that Phe 54 on subunit c (cF54) acts as a hydrophobic barrier preventing protonsfromleaking into the cytoplasm. cF54 also ensures that the c-subunit is positioned correctly in the membrane. While conducting these experiments, we found that an artificial histidine (His) tag on the a-subunit was influencing our results due to its presence in the exit channel and close proximity to cF54. A species of glacial ice worm has been found to possess a His rich sequence in the same location, with possible effects on ATP synthase function. Using an ATP synthase luminescence assay, we tested the effects of removing the His tag from the a-subunit and replacing it with the M.solifugus sequence. Through transgenic mutagenesis of an E. coli plasmid, it has been found that this His rich domain is sufficient to increase ATP synthesis in E. coli. These results show that the effect of His tags on the proteins to which they are attached is not innocuous and can lead to errors in the data that must be accounted for. Our results also show that a change in the positive charge density at the exit channel of the a-subunit can affect both ATP synthesis and hydrolysis.

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