Total Concentration of Ca2 mM

CaM-2Ca

Fig. 11.5 Ca titration of porcine calmodulin and fractional species calculation [25]. (A) Ca-titration for 15 |mM of porcine calmodulin in 50 mM HEPES (pH 7.4, T = 21.5 °C, 90% D2O). Error bars were based on the deviation from two sets of Q-TOF data. The solid curve was the best fit for the average data using the four-parameter model. (B) Fractional species as a function of [Ca2+] for CaM interacting with four Ca2+.

interactions with the first and second calcium ions do not perturb CaM's conformation in any significant way. Therefore, we cannot obtain K1 and K2 from the titration and took these constants from published fluorescence studies that were done under comparable pH and ionic strength [47]. Modeling the titration curve gave and b4, from which we could calculate K3 and K4. The two literature sequential binding constants are: Ki = 2.5 x 105 M-1, K2 = 5.0 x 106 M^1 [47], whereas K3 = 7.1 x 104 M_1, and K4 = 1.1 x 105 M_1 come from PLIMSTEX (Table 11.1); the latter agree with the literature K3 and K4 within a factor of 3. We then calculated the fractional-species of the Ca-bound CaM species, CaM-xCa (x = 0-4) using the macroscopic binding constants K3 and K4 that come from the titration data and the literature values of K1 and K2 (Fig. 11.5B). The binding polynomial shows that apo-CaM (CaM-0Ca) disappears quickly with an increase of total [Ca2+]. CaM-1Ca and CaM-3Ca never become abundant but give way quickly to CaM-2Ca and CaM-4Ca, respectively, substantiating cooperativity in the binding. The major changes in fractional species occur between the formation of CaM-2Ca and CaM-4Ca, which is mirrored by the titration curve, where the greatest difference in exchange also occurs as CaM-2Ca goes to CaM-4Ca. After the fourth Ca2+ is bound, the extent of exchange drops to its lowest level and becomes nearly constant. None of the nonspecific binding of more than four Ca2+ is registered in the titration, indicating that if further binding to Ca2+ occurs, it does not cause any significant conformational changes in the protein. This result demonstrates that PLIMSTEX can determine accurately the intermediate binding species and related binding constants, which are often difficult to obtain by most other mass spectrometry-based methods. It also bypasses any problems caused by nonspecific binding, which is often encountered by direct ESI measurements (complexes involving more than four Ca2+ can be seen by direct ESI).

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