title: Studies of Pseudomonas aeruginosa azurin mutants: cavities in beta-barrel do not affect refolding speed.
abstract: Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.
authors.affiliation: Department of Chemistry, Tulane University, New Orleans, Louisiana 70118, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Pozdnyakova
authors.forename: Jesse
authors.initials: J
authors.lastname: Guidry
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine; D019791:Guanidine; D006639:Histidine; D007700:Kinetics; D008958:Models, Molecular; D010957:Plasmids; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001059:Apoproteins; D001400:Azurin; D006639:Histidine; D019791:Guanidine; D005998:Glycine