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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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Aggregation kinetics monitored by ThT or Congo red binding. A. The aggregation kinetics of the K peptide (˜) monitored by the increase in the ThT fluorescence emission in comparison with those for HEWL (™), the STDY-K peptide (¿) and the W-substituted K peptide GILQINSRG (□). Each peptide sample was incubated at 37°C for up to 14 days in 20 mM glycine-HCl buffer at pH 4, 2, 2 and 2, respectively, prior to mixing with the ThT probe. B. Absorption spectra of Congo red with the K peptides. The peptides were incubated at 37°C in 20 mM glycine-HCl buffer at pH 4 on days 0 (™), 2 (¿), 4 (□) and 7 (˜). Preparations were subjected to these analyses at the concentration of 80 μg/ml as described in Materials and Methods.
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Figure 2: Aggregation kinetics monitored by ThT or Congo red binding. A. The aggregation kinetics of the K peptide (˜) monitored by the increase in the ThT fluorescence emission in comparison with those for HEWL (™), the STDY-K peptide (¿) and the W-substituted K peptide GILQINSRG (□). Each peptide sample was incubated at 37°C for up to 14 days in 20 mM glycine-HCl buffer at pH 4, 2, 2 and 2, respectively, prior to mixing with the ThT probe. B. Absorption spectra of Congo red with the K peptides. The peptides were incubated at 37°C in 20 mM glycine-HCl buffer at pH 4 on days 0 (™), 2 (¿), 4 (□) and 7 (˜). Preparations were subjected to these analyses at the concentration of 80 μg/ml as described in Materials and Methods.

Mentions: The K peptide was dissolved with a small amount of DMSO, and the resulting solution was diluted with each buffer at pH 2.0, 4.0, 7.0 or 9.0 as detailed in Materials and Methods. The prepared preparations were then incubated at 37°C for 1 to 14 days. The K peptide tends to aggregate itself and was dissolved first with DMSO. When DMSO-solubilized K peptide preparations were diluted with high concentration buffer, these produced substantial amounts of amorphous aggregates and obscured the analyses. Accordingly, pH buffers of a comparatively lower concentration (20 mM) were employed. The concentrations of the peptide and DMSO during incubation in the pH buffers were 2 mg/ml and 2%, respectively. The fibrils from K peptide at pH 4 were formed faster, and more were formed, than those at pH 2. The aggregation kinetics was monitored by the increase in the ThT fluorescence emission (Fig. 2A) as a fibrillation index. The results for the K peptide incubated at pH 4 fit a sigmoid curve, indicating the presence of an initial lag phase of about 24 h, followed by a rapid growth phase that reached a plateau on day 7 (solid circles). On the other hand, the K-peptide preparation incubated at pH 2 exhibited a retarded increase of fluorescence intensity reaching a plateau on day 10, while those incubated at pHs 6 and 9 gave hardly any changes (patterns not shown). We inferred that the most effective way to generate ThT-positive products from the K peptide is the incubation at pH 4 for 7 days at 37°C. The rate of increase in fluorescent intensity by the K peptide was higher than that by HEWL itself incubated at pH 2 (open circles). The STDY-K peptide (STDYGILQINSRW, corresponding to the residues 50-62 of HEWL) was confirmed to aggregate at pH 2 more rapidly than the K peptide (diamond patterns), whereas the K-peptide derivative GILQINSRG (the mutant with glycine substituted for tryptophan) gave no signal of aggregation (open squares). To further verify the occurrence of fibrils, Congo red binding to the incubating K peptide samples was performed. As shown in Fig 2B, a time-dependent increase in absorption signal, accompanied by a red shift of the spectral maximum, was observed.


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)

Aggregation kinetics monitored by ThT or Congo red binding. A. The aggregation kinetics of the K peptide (˜) monitored by the increase in the ThT fluorescence emission in comparison with those for HEWL (™), the STDY-K peptide (¿) and the W-substituted K peptide GILQINSRG (□). Each peptide sample was incubated at 37°C for up to 14 days in 20 mM glycine-HCl buffer at pH 4, 2, 2 and 2, respectively, prior to mixing with the ThT probe. B. Absorption spectra of Congo red with the K peptides. The peptides were incubated at 37°C in 20 mM glycine-HCl buffer at pH 4 on days 0 (™), 2 (¿), 4 (□) and 7 (˜). Preparations were subjected to these analyses at the concentration of 80 μg/ml as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Aggregation kinetics monitored by ThT or Congo red binding. A. The aggregation kinetics of the K peptide (˜) monitored by the increase in the ThT fluorescence emission in comparison with those for HEWL (™), the STDY-K peptide (¿) and the W-substituted K peptide GILQINSRG (□). Each peptide sample was incubated at 37°C for up to 14 days in 20 mM glycine-HCl buffer at pH 4, 2, 2 and 2, respectively, prior to mixing with the ThT probe. B. Absorption spectra of Congo red with the K peptides. The peptides were incubated at 37°C in 20 mM glycine-HCl buffer at pH 4 on days 0 (™), 2 (¿), 4 (□) and 7 (˜). Preparations were subjected to these analyses at the concentration of 80 μg/ml as described in Materials and Methods.
Mentions: The K peptide was dissolved with a small amount of DMSO, and the resulting solution was diluted with each buffer at pH 2.0, 4.0, 7.0 or 9.0 as detailed in Materials and Methods. The prepared preparations were then incubated at 37°C for 1 to 14 days. The K peptide tends to aggregate itself and was dissolved first with DMSO. When DMSO-solubilized K peptide preparations were diluted with high concentration buffer, these produced substantial amounts of amorphous aggregates and obscured the analyses. Accordingly, pH buffers of a comparatively lower concentration (20 mM) were employed. The concentrations of the peptide and DMSO during incubation in the pH buffers were 2 mg/ml and 2%, respectively. The fibrils from K peptide at pH 4 were formed faster, and more were formed, than those at pH 2. The aggregation kinetics was monitored by the increase in the ThT fluorescence emission (Fig. 2A) as a fibrillation index. The results for the K peptide incubated at pH 4 fit a sigmoid curve, indicating the presence of an initial lag phase of about 24 h, followed by a rapid growth phase that reached a plateau on day 7 (solid circles). On the other hand, the K-peptide preparation incubated at pH 2 exhibited a retarded increase of fluorescence intensity reaching a plateau on day 10, while those incubated at pHs 6 and 9 gave hardly any changes (patterns not shown). We inferred that the most effective way to generate ThT-positive products from the K peptide is the incubation at pH 4 for 7 days at 37°C. The rate of increase in fluorescent intensity by the K peptide was higher than that by HEWL itself incubated at pH 2 (open circles). The STDY-K peptide (STDYGILQINSRW, corresponding to the residues 50-62 of HEWL) was confirmed to aggregate at pH 2 more rapidly than the K peptide (diamond patterns), whereas the K-peptide derivative GILQINSRG (the mutant with glycine substituted for tryptophan) gave no signal of aggregation (open squares). To further verify the occurrence of fibrils, Congo red binding to the incubating K peptide samples was performed. As shown in Fig 2B, a time-dependent increase in absorption signal, accompanied by a red shift of the spectral maximum, was observed.

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