Figure S2.

Endophilin A1 does not bind Ca²⁺. (A) Purification of recombinant full-length EEN1 expressed in E. coli. Affinity-purified His-tagged EEN1 was supplied with 5 mM EDTA and loaded into Superdex 200 16/300 size exclusion chromatography with buffer (20 mM Tris-Cl, pH 7.0, and 150 mM NaCl). The peak fractions for EEN1 were collected and analyzed by SDS-PAGE. EEN1 displays as monomer and presents negligible degradation without affecting its function. (B) ITC analysis of 1 mM CaCl₂ and 30 µM calmodulin. The raw data were fitted to sequential binding site model. The molar ratio of Ca²⁺ ions (II) to calmodulin is 4, as previously indicated. Ca²⁺ ions (II) appear to bind to calmodulin with dissociation constants (K_d) of 2.26 ± 0.31 µM, 9.08 ± 1.25 µM, 55.1 ± 11.4 µM, and 2.60 ± 2.05 mM, respectively. Each binding site reveals enthalpy changes (ΔH) of 0.22 ± 0.06 kcal/mol 3.31 ± 0.76 kcal/mol, −1.62 ± 0.14 kcal/mol, and 48.1 ± 4.3 kcal/mol, respectively. DP, power differential. (C) ITC analysis of 1 mM CaCl₂, 40 µM EEN1 (left panels) and 3 mM CaCl₂, 100 µM EEN1 (right panels). No significant binding was detected. (D and E) Tryptophan fluorescence of Synaptotagmin-1 C2B (positive control) and EEN1 in the presence of CaCl₂. Shown are tryptophan fluorescence spectra (310–400 nm) of Synaptagmin-1 C2B (D) and EEN1 (E) respectively with an excitation wavelength of 280 nm. Right panels are enlarged views of the emission peaks in the left panels. The tryptophan fluorescence of Synaptagmin-1 C2B increases with the addition of CaCl₂ and saturates at 6 mM final concentration. In contrast, the tryptophan fluorescence of EEN1 fluctuates irregularly with the addition of CaCl₂.