Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix. Issue 19 (25th September 2015)

• Record Type: Journal Article
• Authors: Konuma, Tsuyoshi; Sakurai, Kazumasa; Yagi, Masanori; Goto, Yuji; Fujisawa, Tetsuro; Takahashi, Satoshi
• Is Part Of: Journal of molecular biology. Volume 427:Issue 19(2015:Oct. 01)
• Page Start: 3158
• Page End: 3165
• Publication Date: 2015-09-25
• Subjects: SF stopped-flow -- CF continuous-flow -- βLG β-lactoglobulin -- TFE 2, 2, 2-trifluoroethanol -- SAXS small-angle X-ray scattering -- H/D hydrogen/deuterium; continuous flow rapid mixing -- submillisecond process -- small-angle X-ray scattering -- circular dichroism -- 2, 2, 2-trifluoroethanol; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.07.018
• Languages: English
• ISSNs: 0022-2836

Abstract

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 200 μs. 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. Graphical Abstract: Highlights: Folding of βLG involves intermediates with non-native α-helix. Submillisecond dynamics of βLG folding was observed with CD and SAXS. Initial collapse occurs irrespective of the contents of non-native α-helices. Non-native helix around strand A might facilitate long-range contact formation.