Author/Editor     Žerovnik, E; Virden, R; Jerala, R; Kroon-Žitko, L; Turk, V; Waltho, JP
Title     Difference in the effects of TFE on the folding pathways of human stefins A and B
Type     članek
Source     Proteins
Vol. and No.     Letnik 36, št. 2
Publication year     1999
Volume     str. 205-16
Language     eng
Abstract     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. (Abstract truncated at 2000 characters)
Descriptors     CYSTATINS
CYSTEINE PROTEINASE INHIBITORS
PROTEIN FOLDING
TRIFLUOROETHANOL
AMINO ACID SEQUENCE
CIRCULAR DICHROISM
DOSE-RESPONSE RELATIONSHIP, DRUG
FLUORESCENCE
GUANIDINES
HYDROGEN-ION CONCENTRATION
KINETICS
NUCLEAR MAGNETIC RESONANCE
MOLECULAR SEQUENCE DATA
PROTEIN CONFORMATION
PROTEIN DENATURATION
TITRIMETRY
TYROSINE
ULTRAVIOLET RAYS