Structure?self-assembly And The Nanofibrils Formation Of Oat Protein

The protein nanofibrils generally thought to be associated with more than 20 diseases ,including several neurodegenerative diseases, such as Parkinson's and Alzheimer's and the food protein fibrils could be used as thickening ingredients in low-calorie products, for example, as replacements for polysaccharide-based ingredients or meat replacement products and high protein content foods,also they can be used to accelerate the gel formation and then low the cost of the gel; This study will play a positive role in promoting development of the diseases treatment and health foods.The amino acid compositions, secondary structure, and self-assembly of the oat protein isolate and oat globulin were studied in this dissertation, and the nanofibrils formation of them were further investigated in the nanoscale; The effect of modification of the protein on nanofibrils formation was also researched after the the water soluble compound PEG-OPI being synthesized using hydrophilic polyethylene glycol monomethyl ether (PEG) substitutions.The amino acid compositions, secondary structure, and self-assembly of oat protein isolate (OPI), which was purified from the high-protein Chinese oat, have been investigated by using a combination of amino acid analysis, Fourier transform infrared spectroscopy (FTIR), and tapping mode atomic force microscopy (TP-AFM). OPI, with molecular weights ranging from 14.0 kDa to 66.0 kDa, was rich in essential amino acids and contained 24.7% glutamic acid and 8.1% leucine. The amino acid contents of OPI are 4.5?8.7 times higher than those of oat flour. The secondary structures of OPI have been quantified by the deconvolution of the amide I band of the FTIR spectrum of OPI, which were found to contain approximately 7%?-turn, 19%?-helix, and 74%?-sheet. Tapping mode AFM results further suggest that the oat protein isolate has two major types of shapes, ellipsoidal and disk-like. At protein concentrations below 0.5 mg/mL, most of the OPI molecules are in the isolated form. However, when the concentration of OPI reaches 1.0 mg/mL, some of the OPI molecules self-assembled into large and heterogeneous protein aggregates.The physicochemical and conformational properties, including amino acid composition, subunits, secondary structures, the size and shapes of the individual oat globulin molecule or protein aggregates were evaluated in this study by FTIR, tapping mode AFM, TEM and Dynamic laser light-scattering. Oat globulin, whose molecular weights range from 10.0 KDa to 70.0 KDa, were rich in essential amino acids, and consist of Glutamic acid 24.20% andArginine 8.96%. The amino acid content of oat globulin is 3.78?9.60 times higher than that of oat flour. The secondary structures have been quantified by the deconvolution of the amide I band of the FTIR spectrum of oat globulin, which were found to contain approximately 8.5%?-turn, 10.2%?-helix, and 81.3%?-sheet, which was a bit different from the result of CD spectrum 12.9 %?-helix, 32.8 %?-strand, 25.7%?-turn , and 28.2% random coil, the main difference was the?-turn and random coil.Tapping mode AFM results and TEM further suggest that the oat globulin has major type of global shape. At protein concentrations below 0.1 mg/mL, most of oat globulin molecules are in the isolated form. However, when the concentration of the protein reaches 0.5 mg/mL, some of the molecules self-assembled into large and heterogeneous protein aggregates. Dynamic laser light-scattering showed that the average radius of the oat globulin was about 39.8nm by simple fit with a range from 6nm-200nm, however,the number or mass weighted showed that the radius of about 6nm took up over 90% of the total, the big radius ones belonged to the aggregations,the result is consist with the AFM and TEM.We demonstrate that oat globulin is capable of forming regular elongated fibrils with flat ribbon structure about 4~16nm in height and 0~10?m in length at condition of 24 h ,85°C, protein concentration 1-10 mg/ml,pH 2 as judged from transmission electron microscopy (TEM), atomic force microscopy (AFM), binding of thioflavin T (Th T) and Congo Red dyes, and circular dichroism spectroscopy, it shows that The fibril formation could be greatly influenced by the protein concentration, heating time. Significant protein fibril aggregation occurs when the oat protein concentration increases to 5mg/ml from 2 mg/ml.The morphology of the formed fibrils was closely dependent upon heating time from 0 to 24h. The diameters of the fibrils formed at various times were similar, but the mean contour length progressively increased with heating time. The ThT maximum fluorescence also progressively increased with heating time.The heating process caused remarkable changes in secondary, tertiary, and quaternary conformations of the oat globulin. Gel electrophoresis analysis indicated that heating disrupted the polypeptides of oat globulin, leading to the formation of fragments with lower molecular mass (e.g., <20 kDa after 12h). This appears to be the first direct observation of nanofibrils from oat globulin. The viscosity resulted in a significantly increase in compared to the unheated sample after heating.The morphology fabrication and conformational characteristics of multistranded amyloid-like fibrils as a function of heating time of oat protein isolates (OPI ) were demonstrated by combining of AFM, TEM analysis and DLS techniques, as well as circular dichroism spectroscopy (CD) and SDS-PAGE, as well as binding of thioflavin T (Th T). Results shows that Polydisperse semiflexible nanofibrils with almost the same width can be induced by heating at pH 2.0 from oat protein isolates, and it is suggested that short protofilaments and isolated molecules can touched and aligned with others to developed into a longer contour length ones; Ionic strengths (0–150 mM) had a significant effect on the fibrils formation, the contour length of the stranded aggregates markedly decreased with ionic strength increasing, while the thickness of the strands was nearly unchanged. At ionic strength of 50 mM or above, the stranded aggregates gradually became clustered.A new protein-based water soluble compound PEG-OPI, has been synthesized using hydrophilic polyethylene glycol monomethyl ether (PEG) substitutions. The success of synthesis was confirmed by FT-IR and 1H NMR spectroscopy. In comparison with OPI, the FT-IR spectrum of MPEG-OPI presented new absorption peaks at ~1105 cm-1, which was assigned to ether groups, as well as ~843, 950, and 2886 cm-1 peaks, which arose from MPEG segmen, peaks corresponding to -COCH2CH2CO- group of MPEG-OPI molecule appeared at 3.598,3.651 ppm on its 1H NMR spectrum, the sharp single-peak signal at 3.329 ppm was assigned to -OCH3 of MPEG segments,the modification inhibited the formation of the fibrils as the?-turn structure decreased.