Abstract
The macroscopic Ca(2+)-binding constants of bovine calmodulin have been determined from titrations with Ca2+ in the presence of the chromophoric chelator 5,5'-Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KCl. Identical experiments have also been performed for tryptic fragments comprising the N-terminal and C-terminal domains of calmodulin. These measurements indicate that the separated globular domains retain the Ca2+ binding properties that they have in the intact molecule. The Ca2+ affinity is 6-fold higher for the C-terminal domain than for the N-terminal domain. The salt effect on the free energy of binding two Ca2+ ions is 20 and 21 kJ. mol-1 for the N- and C-terminal domain, respectively, comparing 0 and 150 mM KCl. Positive cooperativity of Ca2+ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains. In the N-terminal domain, the cooperativity amounts to 3 kJ.mol-1 at low ionic strength and greater than or equal to 10 kJ.mol-1 at 0.15 M KCl. For the C-terminal domain, the corresponding figures are 9 +/- 2 kJ.mol-1 and greater than or equal to 10 kJ.mol-1. Two-dimensional 1H NMR studies of the fragments show that potassium binding does not alter the protein conformation.
Highlights
Themacroscopic Ca2+-bindingconstants of bovine calmodulin have been determined from titrations with Ca2+in the presence of the chromophoricchelator 5,5’Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KC1
The crystal form of Ca2+-loadedcalmodulin (Babu et al, 1985, 1988; Kretsinger and Weissman, 1986) resembles a dumbbell with two globular domains separated by a central helix
Small angle x-ray scattering studies (Heidorn and Trewhella, 1988) indicate that calmodulin in solution folds into two separate globular lobes, which on average are closer together than in the crystal state, owing to flexibility in the interconnecting helix
Summary
From Physical Chemistry 2, Lund Uniuersity, Chemical Centre, S-221 00 Lund, Sweden. Themacroscopic Ca2+-bindingconstants of bovine calmodulin have been determined from titrations with Ca2+in the presence of the chromophoricchelator 5,5’Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KC1. In our experi- lator 5,5'-Br,BAPTA (and intactcalmodulin, TRIG, or TR2C, ence, no currently used method for protein concentration determination provides values that are more accurate than theexperimental Ca2+titration data itself (in the case of strong Ca2+binding). This validates the use of F as anadjustable parameter (cf below).For each set of variable parameters, the Newton-Raphson method was used to solve for the free Caz+ concentration, Y , a t each titration point, i, respectively) are shown in Fig. lA together with thecurves of best fit. When all variable parameters (including the protein concentration) wereallowed to adjust their values, the error square sum of the optimal fit was 10-fold higher than for the case of four high affinity sites.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
