FIG 6.

eEF2K H80/H87/H94 are important for CaM binding to eEF2K under low pH. (A) CaM ELISA using the eEF2K(76-95) peptide based on wild-type eEF2K or H80A/H87A/H94A (H3A), H80K/H87K/H94K (H3K), and W85G mutant peptides. Results are expressed as means ± SE from 3 independent experiments. P values were obtained by two-way ANOVA followed by Dunnett's test. *, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001. (B) Calorimetric titrations of wild-type CaM with the eEF2K peptides (78–100) (WT, H80A/H87A/H94A [H3A], and H80K/H87K/H94K [H3K]) at pH 6.8 and 7.5. These experiments were performed at 25°C in 20 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) 150 mM KCl, and 10 mM CaCl₂ at either pH 6.8 or pH 7.5 as described in Materials and Methods. In all cases, the upper graph represents the original titration curve while the lower graph shows the binding isotherm obtained from the experiments shown in the top graph (these data are related to those given in Table 1).