Probing the influence on folding behavior of structurally conserved core residues in P. aeruginosa apo-azurin.
The effects on folding kinetics and equilibrium stability of core mutations in the apo-mutant C112S of azurin from Pseudomonas aeruginosa were studied. A number of conserved residues within the cupredoxin family were recognized by sequential alignment as constituting a common hydrophobic core: I7, F15, L33, W48, F110, L50, V95, and V31. Of these, I7, V31, L33, and L50 were mutated for the purpose of obtaining information on the transition state and a potential folding nucleus. In addition, residue V5 in the immediate vicinity of the common core, as well as T52, separate from the core, were mutated as controls. All mutants exhibited a nonlinear dependence of activation free energy of folding on denaturant concentration, although the refolding kinetics of the V31A/C112S mutant indicated that the V31A mutation destabilizes the transition state enough to allow folding via a parallel transition state ensemble. Phi-values could be calculated for three of the six mutants, V31A/C112S, L33A/C112S, and L50A/C112S, and the fractional values of 0.63, 0.33, and 0.50 (respectively) obtained at 0.5 M GdmCl suggest that these residues are important for stabilizing the transition state. Furthermore, a linear dependence of ln k(obs)(H2O) on DeltaG(U-N)(H2O) of the core mutations and the putative involvement of ground-state effects suggest the presence of native-like residual interactions in the denatured state that bias this ensemble toward a folding-competent state.
Engman
K Cecilia
KC
Department of Chemistry, Göteborg University, Box 465, SE 405 30 Göteborg, Sweden.
Sandberg
Anders
A
Leckner
Johan
J
Karlsson
B Göran
BG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D016415:Sequence Alignment; D013816:Thermodynamics
D001059:Apoproteins; C038778:apoazurin; C050107:cupredoxin; D001400:Azurin