title: Unfolding studies of the cysteine protease baupain, a papain-like enzyme from leaves of Bauhinia forficata: effect of pH, guanidine hydrochloride and temperature.
abstract: Baupain belongs to the α+β class of proteins with a secondary structure-content of 44% α-helix, 16% β-sheet and 12% β-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl)-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. The unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7±0.2 M and 5.0±0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 °C). Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0) and completely unfolded (6.0 M GdnHCl) state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily.
authors.forename: Rosemeire A
authors.initials: RA
authors.lastname: Silva-Lucca
authors.forename: Sheila S
authors.initials: SS
authors.lastname: Andrade
authors.forename: Rodrigo Silva
authors.initials: RS
authors.lastname: Ferreira
authors.forename: Misako U
authors.initials: MU
authors.lastname: Sampaio
authors.affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo 04044-020, SP, Brazil. olivaml.bioq@epm.br.
authors.forename: Maria Luiza V
authors.initials: ML
authors.lastname: Oliva
mesh_terms: D036481:Bauhinia / Q000737:chemistry*; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D010206:Papain / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D058767:Protein Unfolding / Q000187:drug effects; D013696:Temperature; D013816:Thermodynamics
chemical_list: D010206:Papain; D019791:Guanidine