Selective degradation of oxidized calmodulin by the 20 S proteasome

We have investigated the mechanisms that target oxidized calmodulin for deg radation by the proteasome, After methionine oxidation within calmodulin, r ates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragm ents released from the proteasome. Oxidized calmodulin is initially degrade d into large proteolytic fragments that are released from the proteasome an d subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the s elective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. Th ere is a linear correlation between decreases in secondary structure and th e rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in alpha -hel ical content and rates of degradation. In contrast, alterations in the surf ace hydrophobicity of oxidized calmodulin do not alter the rate of degradat ion by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensi tive sites in oxidized calmodulin that are cleaved by the proteasome in a n onprocessive manner.