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The geometrical model introduced previously by the authors has been extended quantitatively to document changes in angular correlations between and among residues as azurin unfolds. In the early stages of denaturation, these changes are found to be more pronounced than changes in the spatial displacement of residues, a result that is also found for residues acting in concert, viz., α-helices, β-sheet residues and residues in ‘turning regions.’ Our analysis leads to a picture of the large-scale motion of the polypeptide chain as azurin denatures. Flanking a central ‘ribbon’ of residues whose orientation remains essentially invariant, we find that in the early stages of unfolding, left- and right-hand ‘wings’ adjacent to this stationary scaffolding pivot counterclockwise, while smaller regions on opposing ends of the β-barrel pivot clockwise. As spatial constraints characterising the native state are further relaxed, our calculations show that some regions reverse their orientational motion, reflecting the enhanced flexibility of the polypeptide chain in the denatured state. [ABSTRACT FROM AUTHOR]

We have designed ruthenium-modified Pseudomonas aeruginosa azurins that incorporate 3-nitrotyrosine (NO2YOH) between Ru(2,2′-bipyridine)2(imidazole)(histidine) and Cu redox centers in electron transfer (ET) pathways. We investigated the structures and reactivities of three different systems: RuH107NO2YOH109, RuH124NO2YOH122, and RuH126NO2YOH122. RuH107NO2YOH109, unlabeled H124NO2YOH122, and unlabeled H126NO2YOH122 were structurally characterized. The pKa's of NO2YOH at positions 122 and 109 are 7.2 and 6.0, respectively. Reduction potentials of 3-nitrotyrosinate (NO2YO-)-modified azurins were estimated from cyclic and differential pulse voltammetry data: oxidation of NO2YO-122 occurs near 1.1 versus NHE; oxidation of NO2YO-109 is near 1.2 V. Our analysis of transient optical spectroscopic experiments indicates that hopping via NO2YO- enhances CuI oxidation rates over single-step ET by factors of 32 (RuH107NO2YO-109), 46 (RuH126NO2YO-122), and 13 (RuH124NO2YO-122). [ABSTRACT FROM AUTHOR]

We investigate the stability to structural perturbation ofPseudomonas aeruginosaazurin using a previously developed geometric model. Our analysis considers Ru(2,2′,6′,2″-terpyridine)(1,10-phenanthroline)(His83)-labelled wild-type azurin and five variants with mutations to Cu-ligating residues. We find that in the early stages of unfolding, the β-strands exhibit the most structural stability. The conserved residues comprising the hydrophobic core are dislocated only after nearly complete unfolding of the β-barrel. Attachment of the Ru-complex at His83 does not destabilize the protein fold, despite causing some degree of structural rearrangement. Replacing the Cys112 and/or Met121 Cu ligands does not affect the conformational integrity of the protein. Notably, these results are in accord with experimental evidence, as well as molecular dynamics simulations of the denaturation of azurin. [ABSTRACT FROM PUBLISHER]

The author cites two studies that demonstrate how reduction potentials in proteins can be rationally tuned using primarily outer-sphere mutations. Topics discussed include the use of azurin as a framework that can be rationally modify to shift reduction potentials of the embedded metal site, the results of a study that tuned reduction potentials in azurin with mutations to onley three residues, and the design of modified proteins relevant to energy and the environment.