Hydrophobic peptides affect binding of calmodulin and Ca2+ as explored by H/D amide exchange and mass spectrometry

Justin B. Sperry, Richard Y.C. Huang, Mei M. Zhu, Don L. Rempel, Michael L. Gross

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


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 Ca2+, the extent of H/DX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca2+-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 Ca2+. Titration of the protein using PLIMSTEX provides the Ca2+-to-calmodulin binding affinity, which agrees well with previously reported values. The affinities of calmodulin to Ca2+ 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 resulting titration data 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.

Original languageEnglish
Pages (from-to)85-92
Number of pages8
JournalInternational Journal of Mass Spectrometry
Issue number1-3
StatePublished - Apr 30 2011


  • Calmodulin
  • Hydrogen/deuterium exchange
  • LC-MS
  • Mastoparan
  • Melittin
  • Myosin light-chain kinase (MLCK)


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