Folding intermediates of a model three-helix bundle protein. Pressure and cold denaturation studies.
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.
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-590, Brazil.
A
A
Chapeaurouge
J S
JS
Johansson
S T
ST
Ferreira
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003080:Cold Temperature; D005453:Fluorescence; D008969:Molecular Sequence Data; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics