The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate

Andrew P. Capaldi, Stuart J. Ferguson, Sheena E. Radford

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40 Scopus citations


Folding of the 123 amino acid residue Greek key protein apo-pseudoazurin from Thiosphaera pantotropha has been examined using stopped-flow circular dichroism in 0.5 M Na2SO4 at pH 7.0 and 15°C. The data show that the protein folds from the unfolded state with all eight proline residues in their native isomers (seven trans and one cis) to an intermediate within the dead-time of the stopped-flow mixing (50 ms). The urea dependence of the rates of folding and unfolding of the protein were also determined. The ratio of the folding rate to the unfolding rate (extrapolated into water) is several orders of magnitude too small to account for the equilibrium stability of the protein, consistent with the population of an intermediate. Despite this, the logarithm of the rate of folding versus denaturant concentration is linear. These data can be rationalised by the population of an intermediate under all refolding conditions. Accordingly, kinetic and equilibrium measurements were combined to fit the chevron plot to an on-pathway model (U⇆I⇆N). The fit shows that apo-pseudoazurin rapidly forms a compact species that is stabilised by 25 kJ/mol before folding to the native state at a rate of 2 s-1. Although the data can also be fitted to an off-pathway model (I⇆U⇆N), the resulting kinetic parameters indicate that the protein would have to fold to the native state at a rate of 86,000 s-1 (a time constant of only 12 μs). Similarly, models in which this intermediate is bypassed also lead to unreasonably fast refolding rates. Thus, the intermediate populated during the refolding of apo-pseudoazurin appears to be obligate and on the folding pathway. We suggest, based on this study and others, that some intermediates play a critical role in limiting the search to the native state.

Original languageEnglish (US)
Pages (from-to)1621-1632
Number of pages12
JournalJournal of Molecular Biology
Issue number5
StatePublished - Mar 12 1999


  • Folding intermediate
  • Greek key
  • On-pathway
  • Proline isomerism
  • Pseudoazurin

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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