Abstract

ATP synthase (ATPase), 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 translocated through FO generate torque to drive F1 to synthesize ATP from ADP and Pi. Previous studies in Escherichia coli (E. coli) ATP synthase showed that proton translocation occurs at the subunit a/c interface (located in FO) and that some amino acid residues in this region are important for function. We are attempting to elucidate what chemical properties are essential for functionality of the isoleucine at position 55 (cI55), which is located on the second transmembrane helix (TMH2) of subunit c. Changes in the side chain were imposed using site directed mutagenesis via polymerase chain reactions (PCR) or through chemical modifications with methanethiosulfonate reagents. ATP-driven H+ pumping activity was observed using fluorescence spectroscopy. So far, replacing isoleucine with glycine, alanine, valine, leucine, and phenylalanine (cI55G, cI55A, cI55V, cI55L, and cI55F) resulted in H+ pumping that behaves similarly to that of the wild type. Proton permeability was also assayed on mutations cI55G, cI55A, cI55F which closely resemble the activity of wild type. Additionally, chemical modifications that increased the steric bulk of the cysteine mutant by adding methyl, propyl, butyl, and benzyl side chains were unable to restore function. These results suggest that if a hydrophobic residue is at this position, ATP synthase can function, regardless of the bulkiness of the amino acid side chain. Mutations to determine if there is an interdependence of cI55 with the adjacent phenylalanine 54 (cF54) as well as the effect that sulfur has at this position are in progress.

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