The folding of a mostly β-sheet protein, intestinal fatty acid binding protein, was examined over a pH range of 4 to 10 in the presence of urea. At pH values ranging from 5 to 10, folding was reversible at equilibrium by circular dichroism (CD) and fluorescence. No significant concentrations of intermediates accumulated at equilibrium, and the stability of the protein was similar over this range. However, at pH 4 and low concentrations of urea (1 to 3 M) significant time-dependent aggregation occurred. High salt concentrations increased the rate and degree of aggregation. Although higher final concentrations of urea (4 to 6 M) resolubilized these aggregates, the fluorescence and circular dichroism spectra of the protein under these conditions were not those of either the native or the unfolded protein. This state was molten globule-like, showing a more intense β-sheet CD signal and a reduced fluorescence intensity with a redshifted emission wavelength maxima compared to the native protein. Higher concentrations of urea (7 to 8 M) unfolded this molten globule form in a cooperative transition. The kinetics of unfolding and refolding were examined by stopped-flow fluorescence. The mechanism of folding and unfolding did not change over the pH range from 6 to 9, with intermediate states observed during both processes. At pH 10 additional phases were observed during both folding and unfolding. The spectral properties of these kinetic intermediates were not similar to those of the molten globule form at pH 4.0. As such, the equilibrium molten globule observed at low pH and high ionic strength does not appear to be on the folding path for this protein.
All Science Journal Classification (ASJC) codes
- Molecular Biology