Probing Ca2+-Induced Conformational Changes in Porcine Calmodulin by H/D Exchange and ESI-MS:  Effect of Cations and Ionic Strength

• Published in: Biochemistry (Easton) 2003-12, Vol.42 (51), p.15388-15397
• Main Authors: Zhu, Mei M, Rempel, Don L, Zhao, Jiang, Giblin, Daryl E, Gross, Michael L
• Format: Article
• Language: English
• Subjects: Animals; Apoproteins - chemistry; Calcium - chemistry; Calmodulin - chemistry; Cations, Divalent - chemistry; Cations, Monovalent - chemistry; Chromatography, Liquid; Deuterium Exchange Measurement - methods; Kinetics; Lithium - chemistry; Magnesium - chemistry; Osmolar Concentration; Potassium - chemistry; Protein Binding; Protein Conformation; Sodium - chemistry; Spectrometry, Mass, Electrospray Ionization - methods; Swine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi035188o

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 determine the conformational changes, binding stoichiometry, and binding constants for Ca2+ interactions with calmodulin (CaM) under varying conditions of electrolyte identity and ionic strength. The outcome shows that CaM becomes less solvent-accessible and more compact upon Ca2+-binding, as revealed by the PLIMSTEX curve. The formation of CaM−4Ca species is the biggest contributor to the shape of the titration curve, indicating that the formation of this species accounts for the largest conformational change in the stepwise Ca2+ binding. The Ca2+-binding constants, when comparisons permit, agree with those in the literature within a factor of 3. The binding is influenced by ionic strength and the presence of other cations, 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 high ionic strength. 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 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. 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 amide hydrogens to shift from the fast to the slow group. The results taken together not only provide new insight into CaM but also indicate that both PLIMSTEX and kinetic modeling of H/D exchange data may become general methods for probing protein conformations and quantifying protein−ligand interactions.