Figure 2. Analysis of the effect of calcium on structural properties and conformational stability of the full-length human CALR. The following buffers were used in these spectroscopic studies: 2mM HEPES, 200mM NaCl, pH=7.0 supplemented with either 2mM EDTA (Apo), 50μM CaCl₂, or 1000μM CaCl₂.

A. ANS fluorescence spectra measured for the full-length human CALR in the absence calcium (black line) or in the presence of 50μM CaCl₂ (red line), or 1000μM CaCl₂ (green line). Spectra of free ANS (blue line) and CALR completely unfolded by 8M urea (pink line) are shown for comparison. In these experiments, protein concentration was 0.01mg/mL, and ANS concentration was kept at the level of 10μM.

B. Urea-induced unfolding of the apo-form of the full-length human CALR (black circles) and the CALR in the presence of 50μM CaCl₂ (red triangles), or 1000μM CaCl₂ (green squares). Urea-induced unfolding was detected by the characteristic red shift of the intrinsic fluorescence spectrum monitored by the urea-induced changes in the F₃₆₀/F₃₂₀ values, where F₃₆₀ and F₃₂₀ correspond to the fluorescence intensity at 360 and 320nm, respectively. Experiments were performed in triplicate. Plots represents averaged F₃₆₀/F₃₂₀ values. Conformational stability of the full-length protein is progressively increased with the increase in calcium concentration.

C. Normalized intrinsic fluorescence spectra of the full-length CALR in the absence calcium (black line) or in the presence of 50μM CaCl₂ (red line), or 1000μM CaCl₂ (green line), and CALR completely unfolded by 8M urea (pink line). Spectra were normalized, since there is a dramatic increase in their maximal intensity which makes the visual analysis of their position difficult.

D. Effect of the increasing urea concentration on the intrinsic fluorescence spectrum of the apo-CALR. Spectra were measured in the presence of 0.0, 0.82, 1.20, 1.65, 2.15, 2.61, 2.94, 3.44, 4.07, 4.59, 5.25, 5.73, 6.59, 7.21, and 7.97 M urea. Increase in urea concentration causes red shift and increase in fluorescence intensity.

E. Effect of the increasing urea concentration on the intrinsic fluorescence spectrum of the full-length human CALR in the presence of 50μM CaCl₂. Spectra were measured in the presence of 0.00, 0.83, 1.68, 2.25, 2.59, 2.94, 3.41, 3.92, 4.21, 4.91, 5.45, 5.98, 6.21, and 6.49 M urea. Increase in urea concentration causes red shift and increase in fluorescence intensity.

F. Effect of the increasing urea concentration on the intrinsic fluorescence spectrum of the full-length human CALR in the presence of 1000μM CaCl₂. Spectra were measured in the presence of 0.00, 0.97, 1.68, 2.10, 2.52, 2.98, 3.44, 3.87, 4.28, 4.78, 5.27, 5.69, 6.10, 6.53, 6.96, 7.39, 7.83, 8.73, and 9.07 M urea. Increase in urea concentration causes red shift and increase in fluorescence intensity.