Phosphorylation Alters the Interaction of the Arabidopsis Phosphotransfer Protein AHP1 with Its Sensor Kinase ETR1
Purified recombinant ETR1(D659A) or ETR1(D659E) at concentrations from 0.04 µM to 20 µM were added to 8 nM fluorescent AHP1-GFP(S65T) in a medium consisting of 50 mM Tris-HCl pH 7.5, 100 mM potassium chloride, 0.1% (w/v) β-D-dodecylmaltoside and 0.002% (w/v) phenylmethylsulfonyl fluoride. For the fluorescent AHP1 either non-phosphorylatable mutant AHP1(H79A)-GFP(S65T) or mutant AHP1(H79E)-GFP(S65T) mimicking permanent phosphorylation were used. Dissociation constants of the mutant ETR1−AHP1 complexes were calculated from the binding curves by Equation 2. ETR1(D659A) and AHP1(H79E)-GFP(S65T) show a Kd of 3.6 µM (Δ,——), ETR1(D659E) and AHP1(H79A)-GFP(S65T) a Kd of 2.6 µM (▴,——), ETR1(D659E) and AHP1(H79E)-GFP(S65T) a Kd of 16.3 µM (•,——) and ETR1(D659A) and AHP1(H79A)-GFP(S65T) a Kd of 15.4 µM (○,——). The dashed grey curve (—) corresponds to the binding characteristics of wild type ETR1 and wild type AHP1 .