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

Lookup NU author(s): Dr Richard Virden


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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 GuHC1, 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-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 GuHC1, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHC1, 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, pit 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 α-helical intermediate of stefin B is highly structured.

Publication metadata

Author(s): Zerovnik E, Virden R, Jerala R, Kroon-Zitko L, Turk V, Waltho JP

Publication type: Article

Publication status: Published

Journal: Proteins: Structure, Function and Genetics

Year: 1999

Volume: 36

Issue: 2

Pages: 205-216

Print publication date: 01/08/1999

ISSN (print): 0887-3585

ISSN (electronic): 1097-0134

Publisher: John Wiley & Sons, Inc.


DOI: 10.1002/(SICI)1097-0134(19990801)36:2<205::AID-PROT6>3.0.CO;2-4

PubMed id: 10398367


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