On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases.
The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60+/-5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved "burst" phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6s) detected by all the spectroscopic probes, which occurred subsequent to an initial "burst" phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed.
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia. eva.zerovnik@ijs.si
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Virden
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Jerala
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Turk
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Waltho
D055312:Cystatin A; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013057:Spectrum Analysis; D014270:Trifluoroethanol / Q000494:pharmacology*
C524994:CSTB protein, human; D055312:Cystatin A; D015891:Cystatins; D011994:Recombinant Proteins; D014270:Trifluoroethanol; C026407:CSTA protein, human; D055313:Cystatin B