β-Lactoglobulin Self-Assembly: Structural Changes in Early Stages and Disulfide Bonding in Fibrils
| العنوان: | β-Lactoglobulin Self-Assembly: Structural Changes in Early Stages and Disulfide Bonding in Fibrils |
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| المؤلفون: | Harjinder Singh, Gillian E. Norris, Simon M. Loveday, Skelte G. Anema, Trevor S. Loo, Anant Dave, Geoffrey B. Jameson |
| المصدر: | Journal of Agricultural and Food Chemistry. 61:7817-7828 |
| بيانات النشر: | American Chemical Society (ACS), 2013. |
| سنة النشر: | 2013 |
| مصطلحات موضوعية: | Protein Folding, Hot Temperature, Protein Conformation, Electrospray ionization, Amino Acid Motifs, Peptide, Lactoglobulins, macromolecular substances, Fibril, chemistry.chemical_compound, Hydrolysis, Phase (matter), Animals, Centrifugation, Disulfides, Sodium dodecyl sulfate, Polyacrylamide gel electrophoresis, Protein Unfolding, chemistry.chemical_classification, Chromatography, Osmolar Concentration, General Chemistry, Hydrogen-Ion Concentration, Crystallography, chemistry, Cattle, General Agricultural and Biological Sciences |
| الوصف: | 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. |
| تدمد: | 1520-5118 0021-8561 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0b2c24bed3308b1fcde1cb62dedf0696Test https://doi.org/10.1021/jf401084fTest |
| رقم الانضمام: | edsair.doi.dedup.....0b2c24bed3308b1fcde1cb62dedf0696 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15205118 00218561 |
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