Avtor/Urednik		Žerovnik, E; Jerala, R; Kroon-Žitko, L; Turk, V; Lohner, K
Naslov		Characterization of the equilibrium intermediates in acid denaturation of human stefin B
Tip		članek
Vir		Eur J Biochem
Vol. in št.		Letnik 245, št. 2
Leto izdaje		1997
Obseg		str. 364-72
Jezik		eng
Abstrakt		Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N - I(N) - I1 - U, where N is the native state, I(N) is a native-like intermediate, I1 is an acid intermediate state with properties of a molten globule and U is the unfolded state. State 1, exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N2 and intermediates I(N2) and I2, which are also dimeric according to size-exclusion chromatography. The acid intermediate I2 is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 1H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalorimetric measurements were performed under conditions where the acid intermediates are maximally populated (18 degrees C): state I(N) from pH 5.0 to 4.6, 0.03 M salt: state I1 below pH 3.8, 0.42 M salt; and state I1 in equilibrium with I(N) at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states I(N) and I1 were comparable to those of the native state. The enthalpy of unfolding for state I1 could not be determined.
Deskriptorji		CYSTATINS CYSTEINE PROTEINASE INHIBITORS SPECTROMETRY, FLUORESCENCE PROTEIN FOLDING PROTEIN DENATURATION OSMOLAR CONCENTRATION MOLECULAR SEQUENCE DATA NUCLEAR MAGNETIC RESONANCE HYDROGEN-ION CONCENTRATION FLUORESCENT DYES CIRCULAR DICHROISM CHROMATOGRAPHY, GEL CALORIMETRY, DIFFERENTIAL SCANNING ANILINO NAPHTHALENESULFONATES