Abstract
The influence of urea, guanidinium chloride (GdmCl), and granidinium thiocyanate (GdmSCN) on the solution structure of lipophilin was examined by circular dischroism and fluorescence techniques. According to the CD results, lipophilin retained at least 60% of organized secondary structure in 8 M urea and 6 M GdmCl (measurements were not possible in GdmSCN). This partial denaturation was of a complex, irreversible nature, and was not appreciably enhanced by prolonged incubation (8 days), by heating to 70 degrees C, by disulfide bond reduction, or by pH variation in the range pH 1.5 to 11. Fluorescence studies demonstrated that the tryptophan residues were only slightly perturbed by 8 M and 6 M GdmCl and remained well buried to the permeant quenching agent acrylamide. A greater, but still far from complete, disruption of lipophilin was achieved in 6 M GdmSCN, and fluorescence polarization provided evidence for some form of cooperative structural change induced by increasing concentrations of this reagent. Transfer of the protein from 2-chloroethanol, in which the tryptophan residues are fully exposed, into 6 M GdmSCN by dialysis resulted in reburial of the fluorophores owing to development of tertiary structure. The combined evidence suggests that the extraordinary resistance of lipophilin to these denaturants is due to the presence of an impervious hydrophobic core. In lipophilin and some other membrane-associated proteins, extended sequences of apolar residues might provide the nuclei for such structural domains.
Highlights
From the Research Institute, The Hospital for Sick Children, Health Sciences Centre, Hamilton, Ontario Toronto, Ontario M5G 1X8, Canada
An abundance of studies has shown that urea and guanidinium salts are very effective protein denaturants, questions regarding the description of the denatured state and the mechanisms involved are not resolved [27]
The fully denatured state is generally thought to approach a random coil conformation [21], though individual proteins vary in their ease of unfolding, and the idealized state may not be reached without cleavage of disulfide bonds [20]
Summary
From the Research Institute, The Hospital for Sick Children, Health Sciences Centre, Hamilton, Ontario Toronto, Ontario M5G 1X8, Canada. The influence of urea, guanidinium chloride (GdmCl), and guanidinium thiocyanate (GdmSCN) on the solution structure of lipophilin was examined by circular dichroism and fluorescence techniques. According to the CD results, lipophilin retained at least 60% of organized secondary structure in 8 M urea and 6 M GdmCl (measurements were not possible in GdmSCN). This partial denaturation was of a complex, irreversible nature, and was not appreciably enhanced by prolonged incubation (8 days), by heating to 7O”C, by disulfide bond reduction, or by pH variation in the range pH 1.5 to 11. The combined evidence suggests that the extraordinary resistance of lipophilin to these denaturants is due to the presence of an impervious hydrophobic core
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