title: Hydrophobic Peptides Affect Binding of Calmodulin and Ca as Explored by H/D Amide Exchange and Mass Spectrometry.
abstract: Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca(2+) and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high resolution PLIMSTEX (Protein-Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca(2+), the extent of HDX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca(2+)-saturated calmodulin exhibits increased protection when bound to the peptides, forming high affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca(2+). Titration of the protein using PLIMSTEX provides the binding affinity of Ca(2+) to calmodulin within previously reported values. The affinities of calmodulin to Ca(2+) increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The titration data taken in this way show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca(2+)-binding properties of this well-studied protein.
authors.affiliation: Department of Chemistry, Washington University in St. Louis, One Brookings Drive, POBox 1134, St. Louis, MO 63130.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Sperry
authors.forename: Richard Y-C
authors.initials: RY
authors.lastname: Huang
authors.forename: Mei M
authors.initials: MM
authors.lastname: Zhu
authors.forename: Don L
authors.initials: DL
authors.lastname: Rempel
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross