Structural Characterization of Calcium-Dependent Calmodulin-Calmidazolium Binding using Capillary Vibrating...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Native mass spectrometry (nMS) analyzes biomolecules in near-native states.
• Ionization methods are critical for preserving molecular integrity during gas-phase transfer.
• Understanding protein-ligand interactions requires methods that maintain native conformations.

Purpose of the Study:

• To evaluate calcium-dependent interactions between calmodulin and calmidazolium (CDZ) using cVSSI-based nMS and in-droplet HDX-MS.
• To assess the ability of cVSSI to preserve native-like states and resolve stepwise Ca2+ binding.
• To characterize CDZ binding, its effect on protein conformation, and binding affinity.

Main Methods:

• Capillary vibrating sharp-edge spray ionization (cVSSI) coupled with native mass spectrometry (nMS).
• In-droplet hydrogen deuterium exchange mass spectrometry (HDX-MS).
• Analysis of calcium-dependent binding and conformational changes of calmodulin.

Main Results:

• cVSSI demonstrated a narrow charge-state distribution (CSD) with low average charge states, indicating native-like preservation.
• Stepwise Ca2+ binding to calmodulin (1-4 Ca2+ ions) was resolved.
• Calmodulin-calmidazolium binding was observed only upon full Ca2+ loading, stabilizing the protein.
• Binding affinity (Kd) was determined to be 261 ± 29 nM (Langmuir) and 126 ± 17 nM (quadratic).
• HDX-MS showed a 23% reduction in deuterium uptake upon ligand binding, indicating stabilization.

Conclusions:

• cVSSI-based nMS coupled with in-droplet HDX-MS provides a comprehensive platform for studying dynamic, metal-dependent protein-ligand interactions.
• This integrated approach allows simultaneous resolution of metal loading, ligand binding, affinity, and conformational changes.
• The method complements traditional structural techniques by directly probing native-state interactions.