Electrospray ionization mass spectrometry and hydrogen/deuterium exchange for probing the interaction of calmodulin with calcium

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Abstract
The extent of H/D exchange of the protein calmodulin in solution was monitored by mass spectrometry following electrospray ionization (ESI) of the protein. In the absence of Ca2+, approximately 115 protons are exchanged for deuteriums after 60 min. As the calmodulin is titrated with Ca2+, the extent of exchange decreases significantly (i.e., by 24 protons), indicating Ca2+-induced folding of the protein to a tighter, less solvent-accessible form. The extent of H/D exchange ceases to decrease when the amount of added Ca2+ is sufficient to convert greater than 80% of the calmodulin to a form bound by four calcium ions. Lysozyme, a protein of similar molecular weight, does not show a significant decrease in the extent of H/D exchange as it binds to Ca2+, indicating that the changes in H/D exchange for calmodulin reflect tertiary structural change that occur upon binding with Ca2+.