Protein-protein interactions between the subunits of the stator stalk of ATP synthase
Gajadeera, Chathurada S.
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F1Fo-ATP synthase is a ubiquitous enzyme responsible for the bulk of ATP synthesis in living cells. ATP synthesis is driven by a transmembrane proton gradient. ATP synthesis/hydrolysis and proton translocation are coupled by a unique rotational mechanism, making ATP synthase a rotary nanomotor. The stator stalk, which is composed of two copies of b subunit and one copy of δ subunit in Escherichia coli is the least well understood part of ATP synthase. The stator stalk attaches the α3β3 ring, which carries the catalytic nucleotide binding sites, to subunit a, which is involved in H+ translocation. The aim of the project is to explore protein-protein interactions between the subunits of the stator stalk of E. coli. To quantitatively characterize binding of b subunit to the δ subunit, a fluorometric assay was developed in which the δ subunit with an introduced tryptophan residue was titrated with a soluble portion of the b subunit, bsol. A Kd of about 1 μM was found for interactions between C-terminal regions of b and δ subunits with the use of δL168W as a probe. To investigate the region of bsol responsible for interaction with δ, binding of a synthetic peptide representing the C-terminal 21 residues of b subunit (b-C21) and of bsol without its C-terminus (bΔc) were studied. The results demonstrated that bΔc binds specifically to δ with a considerable binding affinity (Kd ≈ 4 μM), accounting for a major portion of the binding energy between δ and bsol. Furthermore, I was able to identify the role of the individual b subunits by constructing a fusion protein between one of the b subunits and δ. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b’ type. It was possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. The full-length b subunit (which is covalently linked to δ in the fusion protein) is responsible for connecting the stator stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b’ type.