Phi-value analysis of apo-azurin folding: comparison between experiment and theory.
Pseudomonas aeruginosa azurin is a 128-residue beta-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (phi) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also demonstrate that careful analysis of side-chain interactions is necessary for appropriate theoretical description of core mutants.
Zong
Chenghang
C
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0371, USA.
Wilson
Corey J
CJ
Shen
Tongye
T
Wolynes
Peter G
PG
Wittung-Stafshede
Pernilla
P
D019943:Amino Acid Substitution; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D001400:Azurin; D019791:Guanidine