Info

Fig. 11.6 Ca titration of 15 mM porcine CaM in three different media (99% D2O) [25]. Line(a): 50 mM HEPES/0.1 M KCl, apparent pH 7.4. Line (b) 50 mM HEPES, apparent pH 7.4. Line (c): 2 mM NH4OAc, apparent pH 7.0. Error bars were based on two sets of LCQ titration data. Solid curves were taken from the four-parameter model and are the best fit for the average data.

Fig. 11.6 Ca titration of 15 mM porcine CaM in three different media (99% D2O) [25]. Line(a): 50 mM HEPES/0.1 M KCl, apparent pH 7.4. Line (b) 50 mM HEPES, apparent pH 7.4. Line (c): 2 mM NH4OAc, apparent pH 7.0. Error bars were based on two sets of LCQ titration data. Solid curves were taken from the four-parameter model and are the best fit for the average data.

The DD4 decreases dramatically from low ionic-strength media (2 mM NH4OAc) to high ionic-strength media (50 mM HEPES with 100 mM KCl). The mid points of the curves shift from low [Ca2+]total to higher [Ca2+]total, indicating that the Ca-binding affinity decreases as ionic strength increases. The Ca-binding affinity (K3 K4) obtained from sequential ligand binding curve fitting, where the ratio of protein:ligand is 1:4, decreases by approximately four orders of magnitude with increases in ionic strength and [K+], in agreement with results from other methods [47, 48]. The binding is influenced by ionic strength and the presence of other cations, although many of these cations do not cause conformational changes in apo CaM. Both Ca2+ and Mg2+ bind to CaM with different affinities, causing different conformational changes. K+, if it does bind, causes no detectable conformational change, and the interactions of Ca2+ with CaM in the presence of Li+, Na+, and K+ occur with similar affinities and associated changes in solvent accessibility. These metal-ion effects point to nonspecific rather than competitive binding of alkali metal ions, as was discussed in a full research article [25].

Was this article helpful?

0 0

Post a comment