Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix

Tsuyoshi Konuma, Kazumasa Sakurai, Masanori Yagi, Yuji Goto, Tetsuro Fujisawa, Satoshi Takahashi

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6 Citations (Scopus)


In the folding of β-lactoglobulin (βLG), a predominantly β-sheet protein, a transient intermediate possessing an excess amount of non-native α-helix is formed within a few milliseconds. To characterize the early folding dynamics of βLG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native α-helix formation within 200 μs. Time-resolved SAXS showed that the radius of gyration (Rg) of the intermediate at 300 μs was 23.3 ± 0.7 Å, indicating a considerable collapse from the unfolded state having Rg of 35.1 ± 7.1 Å. Further compaction to Rg of 21.2 ± 0.3 Å occurred with a time constant of 28 ± 11 ms. Pair distribution functions showed that the intermediate at 300 μs comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of α-helix but similar Rg of 23.0 ± 0.8 Å, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native α-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of βLG.

Original languageEnglish
Pages (from-to)3158-3165
Number of pages8
JournalJournal of Molecular Biology
Issue number19
Publication statusPublished - 2015 Sept 25


  • 2 2 2-trifluoroethanol
  • circular dichroism
  • continuous flow rapid mixing
  • small-angle X-ray scattering
  • submillisecond process

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Molecular Biology


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