Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na(2)SO(4) at 15 degrees C), in which one 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 native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold. Study holds ProTherm entries: 7972, 7973 Extra Details: additive : DTT(1 mM),The PDB,PIR and SWISS-PROT codes are for the protein,Pseudoazurin from THIOSPHAERA PANTOTROPHA as given by the authors tyrosine; biophysical techniques; tertiary packing; long-range,interactions; tryptophan
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:35 p.m.
|Number of data points||3|
|Proteins||Pseudoazurin ; Pseudoazurin|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence ATHEVHMLNKGESGAMVFEPAFVRAEPGDVINFVPTDKSHNVEAIKEILPEGVESFKSKINESYTLTVTEPGLYGVKCTPHFGMGMVGLVQVGDAPENLDAAKTAKMPKKARERMDAELAQVN|