Figure 4.

Degradation of oxidized proteins is triggered by disruption of their structure. (a) Analysis by circular dichroism of the secondary structure of native ^{52, 53}calmodulin, oxidized calmodulin and oxidized calmodulin treated with 0.5 mM CaCl₂. The top panel shows the circular dichroism spectra and the bottom panel shows percentages of the different secondary structures obtained by analyzing the circular dichroism spectra using the Contin-LL program. (b) The left panel illustrates a western blot showing that CaCl₂ treatment of oxidized calmodulin reduced its degradation by 20S immunoproteasomes. These results are representative of three independent experiments. The right panel shows that CaCl₂ did not affect the activity of 20S immunoproteasomes, as measured with fluorogenic substrate Suc-LLVY-AMC. (c) Analysis by circular dichroism of the secondary structure of native hemoglobin and oxidized hemoglobin. The top panel shows the circular dichroism spectra and the bottom panel shows percentages of the different secondary structures. (d) Kinetics of degradation of labeled unoxidized and oxidized hemoglobin. These two forms of hemoglobin were incubated with the 20S IP, and protein degradation was monitored as described in Fig. 3e. Similar results were obtained from seven independent experiments. (e, f) Assessing the level of surface exposed hydrophobic patches in the three forms of calmodulin (e) and in the two forms of hemoglobin (f) using the Nile Red probe. Nile Red was incubated with the different samples and its fluorescence at 630 nm, which increases in a non-polar environment, was measured. Relative fluorescence units (RFU) at 630 nm of each sample was reported to the RFU at 630 nm of the corresponding native protein, and the absolute RFU values at 630 nm of the native proteins are indicated on the histogram. All values are mean + SEM of three independent experiments. Full-length images for (b) are presented in Fig. S12.