Differences in the effects of TFE on the folding pathways of human stefins A and B

✍ Scribed by Eva Žerovnik; Richard Virden; Roman Jerala; Louise Kroon-Žitko; Vito Turk; Jonathan P. Waltho

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 275 KB
• Volume: 36
• Category: Article
• ISSN: 0887-3585
• DOI: 10.1002/(sici)1097-0134(19990801)36:2<205::aid-prot6>3.0.co;2-4

✦ Synopsis

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°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 Ϫ1 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°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 ␣-helical intermediate of stefin B is highly struc-