The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site.
Publication Type:Journal Article
Source:Arch Biochem Biophys, Volume 550-551, p.20-7 (2014)
Keywords:Amino Acid Substitution, Azurin, Bacterial Proteins, Circular Dichroism, Copper, Escherichia coli, Gene Expression, Hydrogen Bonding, Hydrogen-Ion Concentration, Kinetics, Mutagenesis, Site-Directed, Oxidation-Reduction, Paracoccus denitrificans, Protein Binding, Protein Interaction Domains and Motifs, Protein Stability, Protein Structure, Secondary, Recombinant Proteins, Spectrometry, Fluorescence, Tryptophan
<p>The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it is separated by 10.1Å. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pKa value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the β-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence.</p>