Figure 2. Kinetics of holo-RD unfolding upon release of calcium.

Panel (A) EDTA-induced dissociation of calcium from holo-RD followed by tryptophan fluorescence spectroscopy. Two mL of 1 μM of holo-RD in buffer A +2 mM CaCl₂ were rapidly mixed with 1 mL of buffer A +5 mM EDTA: the final concentrations of CaCl₂ and EDTA became 1.33 mM and 1.66 mM respectively, yielding a free calcium concentration below 0.48 μM. The data show that Holo-RD is fully converted into the apo-RD state in less than 10 seconds. Panel A Inset: Fluorescence spectra of holo-RD in buffer A +2 mM CaCl₂ (red) and 20 sec after addition of 5 mM EDTA (green). The spectra are stable on the time scale of the RD experiments. Panel (B) Light scattering analysis of the hydrodynamic radius of RD in the presence of 2 mM CaCl₂ (red) and after addition of 5 mM EDTA (green). QELS data were collected and averaged each 10 seconds on RD (12 μM) equilibrated in buffer A +2 mM CaCl₂. EDTA was added at time t = 0 to a final concentration of 5 mM (i.e. residual free calcium below 20 nM), and after 10 sec of mixing, QELS data were similarly collected. The average hydrodynamic radii of holo-RD and apo-RD (open circles with error bars) are 3.2 ± 0.3 and 7.2 ± 1.2 nm, respectively in agreement with our prior results²⁸,³¹. Panel C: Size exclusion chromatography of RD in the presence or absence of calcium. Samples of 5 μM of Holo-RD (dark tone: in buffer A) or apo-RD (light tone: buffer A +2 mM CaCl₂) were loaded on a Superdex 200 10/300 column equilibrated either in buffer A +2 mM CaCl₂ (red chromatogram) or in buffer A (green chromatogram), at a flow rate is 1 mL/min. The retention volume of holo-RD (14 mL) and apo-RD (10 mL) are characteristics of the folded holo-form and the natively disordered apo-form, respectively²⁸,³⁴. Thus, holo-RD was fully converted to the apo-form during the chromatography time (i.e. within less than 10 minutes).