In the cell, energy is required to carry out all necessary functions to keep the cell alive. The carrier for this necessary energy is known as adenosine triphosphate, ATP. The ATP synthase complex is composed of eight unique proteins in a stoichiometry of ab2c10α3β3γεδ, which make up two units, F0 and F1. Fo is the sector responsible for the proton pumping function that occurs due to the proton gradient, which drives ATP production. The main pathway by which protons are translocated through FO is through the a-c interface. There are many interactions that occur in this interface that are thought to contribute to H+ movement and rotation; previous work has determined that the mutation of the residue arginine 50, found in subunit c of the F0 unit, to cysteine inhibits the proton pumping function of ATP synthase. Other work has suggested that the arginine 50 could be a part of a functionally important salt bridge with glutamate 196 on subunits a. To clarify the role of arginine 50 and its potential partners glutamate 196 and aspartate 92, we prepared various mutations of the three residues, and examined their functionality in various biochemical assays. Thus far in the investigation of this residue, we know that altering the size and pKa of the group alters the proton pumping function. However, the ATP synthesis function still needs to be analyzed. Additionally, the genetic construct will be altered from the original template that contains a Histidine tag on the a-c interface, to test the effects of the presence of this tag on function because, based on other studies, this tag could affect the results from function assays. Thus, the first step will be to move all previously made mutations to the new vector and perform function assays to ensure data reliability.
