Residue-Specific Analysis of Frustration in the Folding Landscape of Repeat β/α Protein Apoflavodoxin

Loren Stagg, Antonios Samiotakis, Dirar Homouz, Margaret S. Cheung, Pernilla Wittung-Stafshede

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13 Scopus citations


Flavodoxin adopts the common repeat β/α topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e., at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, φ{symbol}-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding, while proper alignment on both sides of β-strand 3 is necessary for productive folding.

Original languageBritish English
Pages (from-to)75-89
Number of pages15
JournalJournal of Molecular Biology
Issue number1
StatePublished - 12 Feb 2010


  • folding nuclei
  • intermediate
  • protein folding
  • topological frustration
  • transition state


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