Figure 2.

CaMKII has four exposed cysteines, two of which are in the regulatory domain: Cys273 and Cys290.A, purified CaMKII was incubated with iodoacetamide to label exposed cysteine residues (orange color). After that, CaMKII was denatured and reduced by DTT. Acrylamide was added to label the free cysteines (gray color). The enzyme was incubated with trypsin and the mapping was done on HPLC-MS. The experiment was replicated three times. B, the Cys273 mutant or (C) Cys290 mutant were incubated with iodoacetamide at different pHs as indicated in the figure. The percent of derivatized protein molecules was determined by mass spectrometry. The experiment was replicated three times. The curves were fit by Prism to an allosteric sigmoidal. CaMKII, calcium/calmodulin-dependent protein kinase II.