Quantitative determination of binding affinity of delta-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 delta 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 delta contributes zero binding energy. Mg(2+) 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 delta binding affinity, suggesting the involvement of protonatable side-chains in the binding site.
Weber
Joachim
J
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Wilke-Mounts
Susan
S
Senior
Alan E
AE
D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D006180:Proton-Translocating ATPases; D008274:Magnesium