کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1179543 | 962782 | 2014 | 8 صفحه PDF | دانلود رایگان |

• Refolding of an Entamoeba histolytica protease was achieved using SDS.
• Refolding and activation of ppEhCP-B9 occurs only below CMC.
• Conformational refolding routes with surfactants are proposed.
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 β-enriched structure (ppEhCP-B9β1) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α + β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9α + β1). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9β2) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9α + β2) suggesting that enzymatic activity is gained as slow transformation occurs.
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1844, Issue 7, July 2014, Pages 1299–1306