Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a beta-enriched structure (ppEhCP-B9(beta 1)) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a beta-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an alpha + beta structure, similar to that of papain but without proteolytic activity (ppEhCP-B9(alpha + beta 1)). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a beta-sheet rich structure (ppEhCP-B9(beta 2)) that slowly transforms into a different type of alpha + beta conformation that exhibited proteolytic activity (ppEhCP-B9(alpha +) (beta 2)) suggesting that enzymatic activity is gained as slow transformation OMITS. (C) 2014 Elsevier B.V. All rights reserved.