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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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Quenching of tryptophan fluorescence by acrylamide. The K peptide preparations were incubated as described in the legend to Fig. 5 and carried to the quenching test. A. Fluorescence spectra on day 0 (left) and day 7 (right) of incubation. Spectra stand for 0 (F0), 0.05, 0.1, 0.15, 0.02 and 0.25 M of acrylamide from top to bottom. B, Stern-Volmer plots against the quencher concentration. Ksv constant was calculated from the equation of F0/F=1+Ksv[Q] and the results are discussed in the text.
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Figure 7: Quenching of tryptophan fluorescence by acrylamide. The K peptide preparations were incubated as described in the legend to Fig. 5 and carried to the quenching test. A. Fluorescence spectra on day 0 (left) and day 7 (right) of incubation. Spectra stand for 0 (F0), 0.05, 0.1, 0.15, 0.02 and 0.25 M of acrylamide from top to bottom. B, Stern-Volmer plots against the quencher concentration. Ksv constant was calculated from the equation of F0/F=1+Ksv[Q] and the results are discussed in the text.

Mentions: Acrylamide quenching of tryptophan fluorescence was conducted for the K peptide preparations before and after incubation at pH 4 (Fig. 7A, left and right panels, respectively). Tryptophan fluorescence in the day-0 K peptide was quenched by 62% with 0.05 M of acrylamide, whereas that in the day-7 K peptide was quenched only by 41% under the same conditions as above. These facts seemed to imply that the fibrils-forming K peptide requires more acrylamide for quenching tryptophan fluorescence than that in the intact state. Stern-Volmer plots were drawn for the K peptide preparations (Fig. 7B), and from the data the Ksv (Stern-Volmer constant) values were reduced from 19.3, 18.5 18.1 17.1 and 15.3 for the preparations on days 0, 2, 4, 7 and 14, respectively.


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)

Quenching of tryptophan fluorescence by acrylamide. The K peptide preparations were incubated as described in the legend to Fig. 5 and carried to the quenching test. A. Fluorescence spectra on day 0 (left) and day 7 (right) of incubation. Spectra stand for 0 (F0), 0.05, 0.1, 0.15, 0.02 and 0.25 M of acrylamide from top to bottom. B, Stern-Volmer plots against the quencher concentration. Ksv constant was calculated from the equation of F0/F=1+Ksv[Q] and the results are discussed in the text.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Quenching of tryptophan fluorescence by acrylamide. The K peptide preparations were incubated as described in the legend to Fig. 5 and carried to the quenching test. A. Fluorescence spectra on day 0 (left) and day 7 (right) of incubation. Spectra stand for 0 (F0), 0.05, 0.1, 0.15, 0.02 and 0.25 M of acrylamide from top to bottom. B, Stern-Volmer plots against the quencher concentration. Ksv constant was calculated from the equation of F0/F=1+Ksv[Q] and the results are discussed in the text.
Mentions: Acrylamide quenching of tryptophan fluorescence was conducted for the K peptide preparations before and after incubation at pH 4 (Fig. 7A, left and right panels, respectively). Tryptophan fluorescence in the day-0 K peptide was quenched by 62% with 0.05 M of acrylamide, whereas that in the day-7 K peptide was quenched only by 41% under the same conditions as above. These facts seemed to imply that the fibrils-forming K peptide requires more acrylamide for quenching tryptophan fluorescence than that in the intact state. Stern-Volmer plots were drawn for the K peptide preparations (Fig. 7B), and from the data the Ksv (Stern-Volmer constant) values were reduced from 19.3, 18.5 18.1 17.1 and 15.3 for the preparations on days 0, 2, 4, 7 and 14, respectively.

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