文摘
We applied a new method, "protein-ligand interaction using mass spectrometry, titration,and H/D exchange" (PLIMSTEX) [Zhu, M. M. (2003) J. Am. Chem. Soc. 125, 5252-5253], to determinethe conformational changes, binding stoichiometry, and binding constants for Ca2+ interactions withcalmodulin (CaM) under varying conditions of electrolyte identity and ionic strength. The outcome showsthat CaM becomes less solvent-accessible and more compact upon Ca2+-binding, as revealed by thePLIMSTEX curve. The formation of CaM-4Ca species is the biggest contributor to the shape of thetitration curve, indicating that the formation of this species accounts for the largest conformational changein the stepwise Ca2+ binding. The Ca2+-binding constants, when comparisons permit, agree with those inthe literature within a factor of 3. The binding is influenced by ionic strength and the presence of othercations, although many of these cations do not cause conformational change in apo-CaM. Furthermore,Ca2+-saturated CaM exhibits larger protection and higher Ca2+ affinity in media of low rather than highionic strength. Both Ca2+ and Mg2+ bind to CaM with different affinities, causing different conformationalchanges. K+, if it does bind, causes no detectable conformational change, and interactions of Ca2+ withCaM in the presence of Li+, Na+, and K+ occur with similar affinities and associated changes in solventaccessibility. These metal ion effects point to nonspecific rather than competitive binding of alkali-metalions. The rates of deuterium uptake by the various CaM-xCa species follow a three-group (fast,intermediate, slow), pseudo-first-order kinetics model. Calcium binding causes the number of amidehydrogens to shift from the fast to the slow group. The results taken together not only provide new insightinto CaM but also indicate that both PLIMSTEX and kinetic modeling of H/D exchange data may becomegeneral methods for probing protein conformations and quantifying protein-ligand interactions.
