Crystal structure of the double azurin mutant Cys3Ser/Ser100Pro from Pseudomonas aeruginosa at 1.8 Å resolution: its folding–unfolding energy and unfolding kinetics

Abstract

Azurin is a cupredoxin, which functions as an electron carrier. Its fold is dominated by a β-sheet structure. In the present study, azurin serves as a model system to investigate the importance of a conserved disulphide bond for protein stability and folding/unfolding. For this purpose, we have examined two azurin mutants, the single mutant Cys3Ser, which disrupts azurin’s conserved disulphide bond, and the double mutant Cys3Ser/Ser100Pro, which contains an additional mutation at a site distant from the conserved disulphide. The crystal structure of the azurin double mutant has been determined to 1.8 Å resolution², with a crystallographic R-factor of 17.5% (R_free=20.8%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. Also, the rates of folding and unfolding as determined by CD and fluorescence spectroscopy are almost unchanged. The main difference to wild-type azurin is a destabilisation by ∼20 kJ mol⁻¹, constituting half the total folding energy of the wild-type protein. Thus, the disulphide bond constitutes a vital component in giving azurin its stable fold.