Partially unfolded species populated during equilibrium denaturation of the beta-sheet protein Y74W apo-pseudoazurin.


Abstract

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

Submission Details

ID: kbi8pwAC

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Jones S;Reader JS;Healy M;Capaldi AP;Ashcroft AE;Kalverda AP;Smith DA;Radford SE,Biochemistry (2000) Partially unfolded species populated during equilibrium denaturation of the beta-sheet protein Y74W apo-pseudoazurin. PMID:10801317
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3ERX 2009-10-13 1.25 High-resolution structure of Paracoccus pantotrophus pseudoazurin
4BWT 2014-07-16 1.76 Three-dimensional structure of Paracoccus pantotrophus pseudoazurin at pH 6.5
4BXV 2014-07-30 1.76 Three-dimensional structure of the mutant K109A of Paracoccus pantotrophus pseudoazurin at pH 7.0
4BWU 2014-07-16 1.76 Three-dimensional structure of the K109A mutant of Paracoccus pantotrophus pseudoazurin at pH 5.5
1ADW 1997-05-15 2.5 PSEUDOAZURIN

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.1 Pseudoazurin P80649 AZUP_PARDE
100.0 Pseudoazurin P80401 AZUP_PARPN