| Abstract
| - Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which aunique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, aregion that has been proposed to have an important role in folding. Equilibrium denaturation of Y74Wapo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na2SO4 at 15 °C), in whichone or more partially folded species are populated in 4.3 M urea. Using a variety of biophysical techniques,we show that these species, on average, have lost a substantial portion of the native secondary structure,lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the nativestate as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residualstructure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassingthis region, however, did not contain detectable nonrandom structure, suggesting that long-range interactionsare important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basisof these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates oneor more partially unfolded species that are globally collapsed and retain elements of the native structureinvolving the newly introduced tryptophan residue. We speculate on the role of such intermediates in thegeneration of the complex Greek key fold.
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