Differences in the effects of TFE on the folding pathways of human stefins A and B.
Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.
Zerovnik
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E
Department of Biochemistry & Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia. eva.zerovnik@ijs.si
Virden
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Jerala
R
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Kroon-Zitko
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L
Turk
V
V
Waltho
J P
JP
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D055312:Cystatin A; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D019791:Guanidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D017186:Titrimetry; D014270:Trifluoroethanol / Q000494:pharmacology*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
C524994:CSTB protein, human; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D014443:Tyrosine; D014270:Trifluoroethanol; C026407:CSTA protein, human; D055313:Cystatin B; D019791:Guanidine