Quantitative determination of binding affinity of δ-subunit in Escherichia coli F1-ATPase -: Effects of mutation, Mg2+, and pH on Kd

To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of delta-subunit to Escherichia coli F-1-ATPase. The signal used is that of the natural tryptophan at residue delta28, which is enhanced by 50% upon binding of delta-subunit to alpha(3)beta(3)gammaepsilon complex. K-d for delta binding is 1.4 nM, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain. Only one site for 8 binding was detected. The deltaW28L mutation increased K-d to 4.6 nM, equivalent to a loss of 2.9 kJ/mol binding energy. While this was insufficient to cause detectable functional impairment, it did facilitate preparation of delta-depleted F-1. The alphaG29D mutation reduced K-d to 26 nM, equivalent to a loss of 7.2 kJ/mol binding energy. This mutation did cause serious functional impairment, referable to interruption of binding of delta to F-1. Results with the two mutants illuminate how finely balanced is the stator resistance function. delta' fragment, consisting of residues delta1-134, bound with the same K-d as intact delta, showing that, at least in absence of F-o subunits, the C-terminal domain of 5 contributes zero binding energy. Mg2+ ions had a strong effect on increasing delta binding affinity, supporting the possibility of bridging metal ion involvement in stator function. High pH environment greatly reduced 5 binding affinity, suggesting the involvement of protonatable side-chains in the binding site.