| In this body of work, studies of folding kinetics and thermodynamic stability of Pseudomonas aeruginosa azurin, a copper-binding β-barrel protein, are presented. To uncover the role of the copper cofactor in determining the folding pathway of azurin, interactions of the unfolded protein with copper were investigated using a mutagenic approach and a model-peptide system. Based on the findings, the coordination sphere of copper in the unfolded protein is proposed to involve at least two native-state ligands: histidine-117 and cysteine-112. A small peptide corresponding to the carboxy-terminal part of full-length azurin is found to adopt β-sheet-like structure upon copper binding, Formation of this β-hairpin induced by the copper ion in the unfolded protein was originally suggested as a mechanism for folding nucleation. However, detailed folding-kinetic studies demonstrate that the folding speed of azurin is not increased in the presence of copper. It is concluded that formation of the β-hairpin induced by copper binding is not rate limiting for folding.; The topology of the native state is the primary determinant of the folding rate for azurin, as demonstrated by the study of two active-site azurin mutants. His46Gly and His117Gly azurins, in their apo (without cofactor) forms. The decreases in the thermodynamic stability of the mutants, as compared to wild-type azurin, originate from the acceleration of unfolding, However, the folding rates for the mutants are almost identical to what is observed for wild-type apo-azurin. |
