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Originally published In Press as doi:10.1074/jbc.M009622200 on February 5, 2001

J. Biol. Chem., Vol. 276, Issue 18, 14861-14866, May 4, 2001
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Folding Intermediates of a Model Three-helix Bundle Protein
PRESSURE AND COLD DENATURATION STUDIES*

Alex ChapeaurougeDagger §, Jonas S. Johansson, and Sérgio T. FerreiraDagger ||

From the Dagger  Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-590, Brazil and the  Department of Anesthesia and the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

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 (I1). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I2), 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 I2 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.

* This work was supported by National Institutes of Health Grant GM55876 (to J. S. J.) and by a Howard Hughes Medical Institute International Research Scholar Award (to S. T. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ A visiting professor at the Universidade Federal do Rio de Janeiro.

|| To whom correspondence should be addressed. Tel.: 5521-270-5988; Fax: 5521-270-8647; E-mail: ferreira@bioqmed.ufrj.br.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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