Peptide Models of Folding Initiation Sites of Bovine β-Lactoglobulin: Identification of Nativelike Hydrophobic Interactions Involving G and H Strands

Publisher Website
Google Scholar
Abstract
In an attempt to characterize the early folding events in bovine β-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus β-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (β-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native α-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H β-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal α-helix is necessary for stabilizing the BLG folding core.