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Ca2+-free calmodulin was obtained by exhaustively dialyzing 2 mg protein (c = 4 mg/mL) against 1000 volumes of 2 mM EGTA, 50 mM Tris HCl and 150 mM NaCl (pH = 7.4) at 4 °C in a 0.5 mL Pierce Slide-A-LyzerTM Dialysis Cassette (3.5 K MWCO) for 2 days. As the carboxylic groups of EGTA exhibit strong IR bands in the region 1570-1630 cm-1 that overlap with the amide I' band of proteins (1600-1700 cm-1), EGTA was removed by subsequent dialysis against 1000 volumes MilliQ water at 4 °C for 2 days. In both cases, the dialysis buffer and water, respectively, were exchanged every 9-12 h. Calmodulin concentrationwas determined by measuring UV/Vis absorption at 276 nm with 276 = 3006 M-1 cm-1. The appropriate amount of the protein was ε aliquoted and lyophilized overnight in order to remove H2O, which is necessary for FTIR spectroscopy. All measurements were performed in D2O buffer containing 50 mM Tris HCl, 150 mM NaCl and 10 mM CaCl2 (pD 7.4), except for the apo-calmodulin experiments, in which CaCl2 was lacking in buffer. The S-tertbutylthio (StBu) protection group at the N-terminal cysteine of the synthetic peptide was removed by reduction with tris(2carboxyethyl)phosphine (TCEP) before all experiments.