ATP synthase, a ubiquitous biological nanomachine, is responsible for synthesizing the majority of adenosine triphosphate (ATP) in cells. The process of synthesizing ATP uses a unique rotary mechanism, which involves two motors, F1 and FO where protons get translocated in FO. Cryo-electron microscopy (cryo-EM) maps have given insight into the structure of Escherichia coli (E. coli) ATP synthase; however, they do not explain the intricacies of how protons drive rotation. Previous studies showed that proton translocation occurs at the subunit a/c interface (located in FO) and that some amino acid residues are important for function; among these is isoleucine 55 of subunit c (cI55). We are trying to elucidate what chemical properties are essential for functionality at position 55, which is located on the second transmembrane helix (TMH2) of subunit c. Changes in the side chain will be imposed using site directed mutagenesis and chemical modifications via methanethiosulfonate and functionality observed using fluorescence spectrometry. Replacing isoleucine with alanine (cI55A) resulted in H+ pumping that behaves similarly to that of the wild type. This result leads us to believe that steric bulk is not an essential property at this position, and we are currently looking at the importance of hydrophobicity.
