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

In the folding of beta-lactoglobulin (beta LG), a predominantly beta-sheet protein, a transient intermediate possessing an excess amount of non-native a-helix is formed within a few milliseconds. To characterize the early folding dynamics of beta LG 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 alpha-helix formation within 200 mu s. Time-resolved SAXS showed that the radius of gyration (R-g) of the intermediate at 309 mu s was 23.3 +/- 0.7 angstrom, indicating a considerable collapse from the unfolded state having R-g of 35.1 +/- 7.1 angstrom. Further compaction to R-g of 21.2 +/- 0.3 angstrom occurred with a time constant of 28 +/- 11 ms. Pair distribution functions showed that the intermediate at 300 mu s 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 alpha-helix but similar R-g of 23.0 +/- 0.8 angstrom, 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 alpha-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 beta LG. (C) 2015 Elsevier Ltd. All rights reserved.