[No authors listed]
ATP-dependent proteases unfold their substrates and then refold (via chaperone activity) or degrade (via protease activity) them. The proteases choose between these two activities by selecting their substrates; however, little is known about their substrate selection mechanism. The present study attempts to clarify this mechanism by investigating the role of the Escherichia coli (E. coli) ATP-dependent protease ClpAP. To address this, a reaction system that can measure both chaperone and protease activities simultaneously must be constructed. However, the chaperone activities cannot be evaluated in the presence of protease units. Green fluorescent protein (GFP) is usually used as a model substrate of ClpAP; the fluorescence decrease reflects the degradation of substrates. However, it is difficult to evaluate the chaperone activity of ClpAP using this system, because it cannot distinguish between intact and refolded substrates. Therefore, it is necessary to evaluate the exact unfolding activity while avoiding restoration of substrate spectroscopic characteristics due to chaperone activity. In this study, E. coli Ferredoxin (Fd) was used as a new model substrate for ClpAP to evaluate its unfolding activity. Intact and refolded substrates may be distinguished by the existence of an Fd Fe-S cluster. To verify this hypothesis, the absorption spectrum of Fd complexed with ClpA, the chaperone unit of ClpAP, was measured. A decrease in two peaks derived from the Fe-S cluster was observed, indicating that the Fe-S cluster of Fd was disrupted by the ClpA chaperone. This reaction system should prove useful to evaluate the exact unfolding activity of ATP-dependent proteases.
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