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Analysis of core region from egg white lysozyme forming amyloid fibrils.

Tokunaga Y, Sakakibara Y, Kamada Y, Watanabe K, Sugimoto Y - Int. J. Biol. Sci. (2013)

Bottom Line: The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C.A substantial number of fibrils were generated under this pH condition and incubation period.Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Bioscience The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065 Japan.

ABSTRACT
Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.

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Intrinsic tryptophan fluorescence due to the K peptide. Samples were prepared as described in the legend to Fig. 5 and analyzed for the tryptophan fluorescence emission. Excitation wavelength was set to 295 nm and emission wavelength to 320-450 nm.
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Figure 6: Intrinsic tryptophan fluorescence due to the K peptide. Samples were prepared as described in the legend to Fig. 5 and analyzed for the tryptophan fluorescence emission. Excitation wavelength was set to 295 nm and emission wavelength to 320-450 nm.

Mentions: The K peptide consists of hydrophobic amino acids including tryptophan that is sited at the C-terminal. Therefore, the fluorescence intensity of tryptophan is also likely to mirror the configuration features of this peptide. The K peptide during incubation at pH 4 was analyzed for its intrinsic tryptophan fluorescence spectrum, which increased gradually and attained the maximum on day 7 (Fig. 6). It was around this time when ThT fluorescent reached a plateau and fibrils were clearly observed in the K peptide preparation (see Figs. 2A and 3). Comparable data were obtained in the K-peptide incubated at pHs 2, 7 and 9, although the fluorescence intensity was low in these cases (data not shown).


Analysis of core region from egg white lysozyme forming amyloid fibrils.

Tokunaga Y, Sakakibara Y, Kamada Y, Watanabe K, Sugimoto Y - Int. J. Biol. Sci. (2013)

Intrinsic tryptophan fluorescence due to the K peptide. Samples were prepared as described in the legend to Fig. 5 and analyzed for the tryptophan fluorescence emission. Excitation wavelength was set to 295 nm and emission wavelength to 320-450 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3584918&req=5

Figure 6: Intrinsic tryptophan fluorescence due to the K peptide. Samples were prepared as described in the legend to Fig. 5 and analyzed for the tryptophan fluorescence emission. Excitation wavelength was set to 295 nm and emission wavelength to 320-450 nm.
Mentions: The K peptide consists of hydrophobic amino acids including tryptophan that is sited at the C-terminal. Therefore, the fluorescence intensity of tryptophan is also likely to mirror the configuration features of this peptide. The K peptide during incubation at pH 4 was analyzed for its intrinsic tryptophan fluorescence spectrum, which increased gradually and attained the maximum on day 7 (Fig. 6). It was around this time when ThT fluorescent reached a plateau and fibrils were clearly observed in the K peptide preparation (see Figs. 2A and 3). Comparable data were obtained in the K-peptide incubated at pHs 2, 7 and 9, although the fluorescence intensity was low in these cases (data not shown).

Bottom Line: The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C.A substantial number of fibrils were generated under this pH condition and incubation period.Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Bioscience The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065 Japan.

ABSTRACT
Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.

Show MeSH
Related in: MedlinePlus