TY - JOUR
T1 - Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica
AU - Zamudio-Prieto, Olga
AU - Benítez-Cardoza, Claudia
AU - Arroyo, Rossana
AU - Ortega-López, Jaime
N1 - Funding Information:
This work was supported by CINVESTAV-IPN and CONACyT under grants 49987-Z and 128694 , respectively, (to JOL) and a PhD scholarship 290575 (to OZP). We thank MSc. Claudia Ivonne Flores Pucheta and María Eugenia Zuñiga Trejo for their technical assistance. We especially thank Alejandro Mejia Greene for his assistance with the figures. Thanks to the reviewers for their constructive comments and suggestions.
PY - 2014/7
Y1 - 2014/7
N2 - 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-B9 β1) 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-B9 β2) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9 α + β2) suggesting that enzymatic activity is gained as slow transformation occurs.
AB - 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-B9 β1) 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-B9 β2) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9 α + β2) suggesting that enzymatic activity is gained as slow transformation occurs.
KW - Chemical denaturation
KW - Cysteine protease
KW - Entamoeba histolytica
KW - Protein folding
KW - SDS
UR - http://www.scopus.com/inward/record.url?scp=84899807108&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2014.04.009
DO - 10.1016/j.bbapap.2014.04.009
M3 - Artículo
C2 - 24769005
SN - 1570-9639
VL - 1844
SP - 1299
EP - 1306
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 7
ER -