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.
Highlights
We have previously reported the biochemical characterization of a cysteine protease fromBauhinia forficata leaves, named baupain [1]
Our findings showed that baupain has kininogenase activity [1] and blocks thrombin-induced human platelet aggregation, possibly by unspecific cleavage of protease activated receptors 1 (PAR1) [2]
Cluster analysis showed that baupain belongs to the α+β tertiary structure class, like other cysteine proteases, for example procerain and bromelain [8,11]
Summary
We have previously reported the biochemical characterization of a cysteine protease from. The molten globule state, a non-native compact state of a protein, is typically regarded as a general intermediate in protein folding. This state is characterized by the presence of significant amount of secondary structure, lack of most specific tertiary structures associated with tight packing of side chains, and the presence of a loosely packed hydrophobic core, which is accessible to solvent molecules. We investigated the conformational behavior of baupain in solution as a function of pH, temperature and chemical denaturants. Additional studies about the conformational behavior of similar proteases from different members of the cysteine protease family would be important towards elucidating the folding pathway of this class of proteins
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