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

Biochim Biophys Acta. 2014 Jul;1844(7):1299-306. doi: 10.1016/j.bbapap.2014.04.009. Epub 2014 Apr 24.

Abstract

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.

Keywords: Chemical denaturation; Cysteine protease; Entamoeba histolytica; Protein folding; SDS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Circular Dichroism
  • Cysteine Proteases / chemistry*
  • Cysteine Proteases / isolation & purification
  • Cysteine Proteases / metabolism
  • Detergents / pharmacology*
  • Entamoeba histolytica / enzymology*
  • Micelles
  • Protein Conformation / drug effects*
  • Protein Denaturation / drug effects*
  • Protein Folding / drug effects*
  • Protein Renaturation / drug effects*
  • Recombinant Proteins / metabolism
  • Spectrometry, Fluorescence

Substances

  • Detergents
  • Micelles
  • Recombinant Proteins
  • Cysteine Proteases