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 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-B9(α+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.
Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, Col. San Pedro Zacatenco, Mexico, DF 07360, Mexico. Electronic address: olga_zamudio@hotmail.com.
Olga
O
Zamudio-Prieto
Laboratorio de Investigación Bioquímica, ENMyH-IPN, Guillermo Massieu Helguera No. 239, La Escalera Ticoman, Mexico,DF 07320, Mexico. Electronic address: beni1972uk@gmail.com.
Claudia
C
Benítez-Cardoza
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Col. San Pedro Zacatenco, Mexico, DF 07360, Mexico. Electronic address: rarroyo@cinvestav.mx.
Rossana
R
Arroyo
Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, Col. San Pedro Zacatenco, Mexico, DF 07360, Mexico. Electronic address: jortega@cinvestav.mx.
Jaime
J
Ortega-López
D002942:Circular Dichroism; D057056:Cysteine Proteases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D004748:Entamoeba histolytica / Q000201:enzymology*; D008823:Micelles; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation / Q000187:drug effects*; D017510:Protein Folding / Q000187:drug effects*; D020673:Protein Renaturation / Q000187:drug effects*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D003902:Detergents; D008823:Micelles; D011994:Recombinant Proteins; D057056:Cysteine Proteases
Chemical denaturation; Cysteine protease; Entamoeba histolytica; Protein folding; SDS