Differences in the effects of TFE on the folding pathways of human stefinsA 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 m ol/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 den atured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final condi tions. 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 w as only twice that observed when TFE was absent. As a major point of distin ction from stefin A, the refolding of stefin B in the presence of TFE showe d an overshoot in the ellipticity at 230 nm to a value 10% greater than tha t in the native protein; this signal relaxed slowly (0.01 s(-1)) to the fin al native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturati on in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same fina l conditions exhibited the same rate-limiting step (0.01 s(-1)) but were fa ster 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 intermedi ate of stefin B is highly structured. (C) 1999 Wiley-Liss, Inc.