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Apo-azurin folds via an intermediate that resembles the molten-globule

¹ Department of Chemistry and
² Swedish NMR Centre, Göteborg University, SE 405 30 Göteborg, Sweden
³ Department of Chemistry and Bioscience, Chalmers University of Technology, SE 405 30 Göteborg, Sweden

(RECEIVED May 7, 2004; FINAL REVISION July 1, 2004; ACCEPTED July 2, 2004)

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (G_U–N^H2O) of the C112S and C112A mutants were measured to 36.8 ± 1 kJ mole^–1 and 26.1 ± 1 kJ mole^–1, respectively, and the m-value of the transition to 23.5 ± 0.7 kJ mole^–1 M^–1. The difference in folding free energy (G_U–N^H2O) is largely attributed to the intramolecular hydrogen bonding properties of the serine O in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed G_U–N^H2O. This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.

Keywords: protein folding; intermediate; -sandwich; Greek key; azurin; cupredoxin

Abbreviations: ANS, 1-anilinonaphtalene-8-sulfonate • _TS, Tanford -value for the transition state • _I, Tanford -value for the intermediate state • CD, circular dichroism • [D], denaturant concentration • [D]_50%, mid-point of denaturation • GdmCl, guanidinium chloride • I, intermediate state • N, native state • TS, transition state • U, unfolded state.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04848204.

Reprint requests to: B. Göran Karlsson, Swedish NMR Centre, Göteborg University, Box 465, SE 405 30 Göteborg, Sweden; e-mail: goran{at}nmr.se; fax: +46-31-773-3880.

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