Solvation of the folding-transition state in Pseudomonas aeruginosa azurin is modulated by metal: Solvation of azurin's folding nucleus.
The role of water in protein folding, specifically its presence or not in the transition-state structure, is an unsolved question. There are two common classes of folding-transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (phi) values, and polarized transition states, which instead display distinct substructures with very high phi-values. Apo-and zinc-forms of Pseudomonas aeruginosa azurin both fold in two-state equilibrium and kinetic reactions; while the apo-form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding-transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic-to-alanine phi-value analysis, valine-to-threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding-transition state upon combination with regular phi-values. We find that the transition state for folding of apo-azurin appears completely dry, while that for zinc-azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo-and holo-folding nuclei can be rationalized in terms of the shape of the free-energy barrier.
Wilson
Corey J
CJ
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, USA.
Apiyo
David
D
Wittung-Stafshede
Pernilla
P
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D019791:Guanidine / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism / Q000494:pharmacology*; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry* / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D008670:Metals; D012997:Solvents; D014867:Water; D001400:Azurin; D013912:Threonine; D014633:Valine; D015032:Zinc; D019791:Guanidine; D000409:Alanine