Figure 2.

1D ¹H‐spectra obtained with (A) addition of EDTA to the apo‐CRD, (B) addition of EDTA to the holo‐CRD, and (C) addition of excess EDTA to the holo‐CRD. All spectra were recorded at pH 6.8. EDTA can be used to probe for the presence of Ca²⁺, as the 1D spectra of free and Ca²⁺‐bound EDTA are easily identified. Assignments are mapped onto the EDTA structure, and the pH was kept constant for all spectra. Addition of EDTA to the apo‐CRD (A) produced only free EDTA peaks, confirming that there was no Ca²⁺ present, and that the protein was indeed in the apo‐form. In contrast, addition of EDTA to the holo‐CRD (B) produced only Ca²⁺‐bound EDTA peaks, as the free Ca²⁺ interacts with EDTA. Addition of excess EDTA to the holo‐CRD (C) resulted in the appearance of additional free EDTA peaks.