Bovine serum fetuin is unfolded through a molten globule state.
The reversible heat- and GuHCl-induced unfolding of bovine serum fetuin (BSF) has been studied by differential scanning calorimetry, circular dicroism, tryptophan fluorescence, and size-exclusion chromatography. We show here that thermal unfolding of BSF occurs in two distinct steps corresponding to transitions from the native (N) to an intermediate (I) and from the intermediate to the unfolded state (U). The Nleft and right arrow I transition is highly cooperative and can well be accounted for by a two-state mechanism. The Ileft and right arrow U transition is also cooperative but to a lesser extent than the Nleft and right arrow I transition. CD spectra show that the protein in the I state retains nearly all of the native secondary structure and has a largely disrupted tertiary structure. However, the hydrophobic environment of the single tryptophan residue is not changed, and some compactness is retained in the I state. The structural properties of this intermediate state are apparently characteristic of a molten globule. The GuHCl-induced unfolding is also a two-step process with an I state around 2 M GuHCl. Although the structural features of the denaturant-induced I state are somewhat different from those of the heat-induced I state, the unfolding free energies DeltaG degreesNleft and right arrow I and DeltaG degreesIleft and right arrow U as well as DeltaG degreesNleft and right arrow U obtained from these two methods are comparable. We argue that the observed two-state Nleft and right arrow I transition is due to the melting of the tertiary packing, while leaving quasi-intact the secondary structure and some long-range interactions in the I state. These long-range interactions, together with the secondary structural elements, would be responsible for the observed cooperativity of the Ileft and right arrow U transition.
Wang
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Institute for Structural Biology and Drug Discovery, Virginia Biotechnology Research Park, Richmond 23298, USA.
Lascu
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Giartosio
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D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019791:Guanidine; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D000509:alpha-Fetoproteins / Q000737:chemistry*
D000509:alpha-Fetoproteins; D019791:Guanidine