Electrospray ionization mass spectrometry and hydrogen/deuterium exchange for probing the interaction of calmodulin with calcium.
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 Ca(2+)-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+.
Nemirovskiy
O
O
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Giblin
D E
DE
Gross
M L
ML
D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D003903:Deuterium; D006859:Hydrogen; D013058:Mass Spectrometry; D009113:Muramidase / Q000737:chemistry; D011487:Protein Conformation; D013552:Swine
D002147:Calmodulin; D006859:Hydrogen; D003903:Deuterium; D009113:Muramidase; D002118:Calcium