Abstract
The stability and equilibrium unfolding of a model three-helix bundle protein, alpha(3)-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of alpha(3)-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid). The overall free-energy change for complete unfolding of alpha(3)-1, determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native state is stabilized by -1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I(1)). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I(2)), suggesting a significant contribution of hydrophobic interactions to the stability of alpha(3)-1. The free energy of stabilization of the native-like state relative to I(2) is evaluated to be -2.5 kcal/mol. Bis-ANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of alpha(3)-1. The demonstration of folding intermediates of alpha(3)-1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.
Highlights
The stability and equilibrium unfolding of a model three-helix bundle protein, ␣3-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated
BisANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partiallyfolded states of ␣3-1
Where N and U are the spectral centers of mass of native-like and fully unfolded protein obtained in the absence of denaturant and in the presence of a high concentration of GdnHCl, respectively, p is the spectral center of mass at pressure p, and Q is the ratio of fluorescence quantum yields of unfolded and native-like ␣3-1
Summary
The native state is stabilized by ؊1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I1). BisANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of ␣3-1. Recent simulation studies have suggested the existence of intermediate states during the folding of a small model three-helix bundle protein (9 –12). Our results revealed the existence of partially (un)folded intermediate states of ␣3-1, giving support to the predictions from the above mentioned simulation studies. Bis-ANS binding studies revealed the existence of significant residual hydrophobic structure in the pressure-denatured and especially in the cold-denatured state of ␣3-1, suggesting molten globule-like conformations for these intermediates
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