Folding intermediates of a model three-helix bundle protein - Pressure andcold denaturation studies

The stability and equilibrium unfolding of a model three-helix bundle prote in, alpha (3)-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturi ng agents allowed detection of two partially folded states of alpha (3)-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fl uorescence of the hydrophobic probe bis-ANS (4,4'-dianilino-1,1'-binaphthyl -5,5'-disulfonic acid). The overall free-energy change for complete unfoldi ng 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 f olded pressure-denatured intermediate (I-1). Cold denaturation at high pres sure gives rise to a second partially (un)folded conformation (I-2), sugges ting a significant contribution of hydrophobic interactions to the stabilit y 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 p ressure- and cold denatured states indicates the existence of significant r esidual hydrophobic structure in the partially (un)folded states of alpha ( 3)-1, The demonstration of folding intermediates of alpha (3)-1 lends exper imental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such int ermediates. The results are discussed in terms of the significance of de no vo designed proteins for protein folding studies.