title: Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix.
abstract: 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 200μs. Time-resolved SAXS showed that the radius of gyration (R(g)) of the intermediate at 300 μs was 23.3±0.7 Å, indicating a considerable collapse from the unfolded state having R(g) of 35.1±7.1 Å. Further compaction to R(g) 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 R(g) 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.
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Konuma
authors.affiliation: High Pressure Protein Research Center, Institute of Advanced Technology, Kindai University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Sakurai
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Yagi
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.affiliation: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan; RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Fujisawa
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan. Electronic address: st@tagen.tohoku.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D007782:Lactoglobulins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D053838:Scattering, Small Angle; D014270:Trifluoroethanol / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D007782:Lactoglobulins; D014270:Trifluoroethanol
keywords: 2,2,2-trifluoroethanol; circular dichroism; continuous flow rapid mixing; small-angle X-ray scattering; submillisecond process