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Research Article

Solvation of the folding-transition state in Pseudomonas aeruginosa azurin is modulated by metal

¹ Department of Biochemistry and Cell Biology ² Keck Center for Structural Computational Biology ³ Department of Chemistry, Rice University, Houston, Texas 77251, USA

(RECEIVED September 13, 2005; FINAL REVISION December 14, 2005; ACCEPTED December 18, 2005)

The role of water in protein folding, specifically its presence or not in the transition-state structure, is an unsolved question. There are two common classes of folding-transition states: diffuse transition states, in which almost all side chains have similar, rather low phi () values, and polarized transition states, which instead display distinct substructures with very high -values. Apo- and zinc-forms of Pseudomonas aeruginosa azurin both fold in two-state equilibrium and kinetic reactions; while the apo-form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding-transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic-to-alanine -value analysis, valine-to-threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding-transition state upon combination with regular -values. We find that the transition state for folding of apo-azurin appears completely dry, while that for zinc-azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo- and holo-folding nuclei can be rationalized in terms of the shape of the free-energy barrier.

Keywords: protein structure/folding; conformational changes; stability and mutagenesis; circular dichroism; fluorescence; molecular mechanics/dynamics; kinetics

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051838206

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