Folding and Structure of Azurin - The Influence of a Metal
The structural role of the metal in the protein azurin from Pseudomonas aeruginosa has been a long standing question. Azurin belongs to the cupredoxin family and is a 128 residue beta-barrel protein of greek-key topology. The biological function of azurin is that of an electron carrier, and the electron shuttling is mediated via a redox active copper ligand that is coordinated by the protein. Another structural feature of azurin is an N-terminal disulfide bond that is not conserved within the cupredoxin family.
This thesis contains the near complete NMR assignments, as well as the solution structure of diamagnetic (Cu(I)) azurin. Furthermore, assignments of the contact shifted residues in the paramagnetic (Cu(II)) form and an investigation of the pseudocontact shift contribution is presented.
The investigation of the folding covers different azurin species: metal ligand substitutions, engineered apo-mutants and mutants lacking the disulfide bond. Equilibrium folding investigations show that the metal ion remains bound to the protein in the denatured state, and that azurin is more stable in the oxidized Cu(II) form than in the reduced Cu(I) form. The stabilization is primarily an entropic destabilization of the unfolded state, and is ascribed to a tetragonal metal coordination in the oxidized form that changes to trigonal upon reduction. From kinetic folding measurements, the influence of the metal ion and the disulfide bond on the folding process can be deduced. The folding behavior is significantly different in the apo-form, compared to the metal bound form, changing from a two-state to a three-state process. Removing the disulfide bond only affects the folding and unfolding rates. It is found that neither the metal, nor the disulfide bond, are important for the folding of azurin, but that both significantly stabilize azurin by reducing the unfolding rate.