title: Β-lactoglobulin self-assembly: structural changes in early stages and disulfide bonding in fibrils.
abstract: Bovine β-lactoglobulin (β-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating β-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of β-Lg during the lag phase and the composition of β-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated β-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of β-Lg. The SDS-PAGE profiles of fibrils separated by ultra centrifugation showed that after six hours, the fibrils consisted of a few preferentially accumulated peptides. Two-dimensional SDS-PAGE under reducing and nonreducing conditions showed the presence of disulfide-bonded fragments in the fibrils. The sequences in these peptide bands were characterized by in-gel digestion electrospray ionization (ESI)-MS/MS. The composition of solubilized fibrils was also characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS/MS. Both MS analyses showed that peptides in fibrils were primarily from the N-terminal region, although there was some evidence of peptides from the C-terminal part of the molecule present in the higher molecular weight gel bands. We suggest that although the N-terminal region of β-Lg is almost certainly involved in the formation of the fibrils, other peptide fragments linked through disulfide bonds may also be present in the fibrils during self-assembly.
authors.affiliation: Riddet Institute, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
authors.forename: Anant C
authors.initials: AC
authors.lastname: Dave
authors.forename: Simon M
authors.initials: SM
authors.lastname: Loveday
authors.forename: Skelte G
authors.initials: SG
authors.lastname: Anema
authors.forename: Trevor S
authors.initials: TS
authors.lastname: Loo
authors.forename: Gillian E
authors.initials: GE
authors.lastname: Norris
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Jameson
authors.forename: Harjinder
authors.initials: H
authors.lastname: Singh
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D009994:Osmolar Concentration; D011487:Protein Conformation; D017510:Protein Folding; D058767:Protein Unfolding
chemical_list: D004220:Disulfides; D007782:Lactoglobulins