Crosslinking study and rotational assay of ATP Synthase

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F1Fo-ATP synthase is the enzyme responsible for the synthesis of ATP, by the process of oxidative phosphorylation and photophosphorylation in mitochondria, chloroplasts and bacteria. ATP serves as the primary energy source in the cell. The ATP synthase is a multi subunit enzyme complex that is made up of two sub-complexes F1 and Fo. The proton gradient generated by the electron transfer through the electron transport chain serves as the driving force for the ATP synthesis. The Fo sub complex converts the energy stored in the electrochemical gradient into the rotation of the subunit which causes the rotation of the F1 subunit. The synthesis of ATP is a result of the interactions between the Fo and F1 subunit. F1 acts as ATPase when isolated. The α3β3γ complex isolated from Bacillus PS3 is a stable cysteine less enzyme complex even at high temperatures. The amino acids αG50 and βD67 were present in the catalytic interface between the α and β chains. The amino acids were replaced with cysteine to prepare a double mutant. The amino acids αE84 and βH53 are present in the non catalytic interface. These amino acids were also replaced with cysteine in a second double mutant. The replacement with cysteine enabled crosslinking to occur in the double mutants. The double mutant proteins were then subjected to high temperatures and freeze thaw cycles to check their stability. The ATPase activities at different temperatures of the crosslinked samples were compared with the non crosslinked sample.