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Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

Mira H, Vilar M, Esteve V, Martinell M, Kogan MJ, Giralt E, Salom D, Mingarro I, Peñarrubia L, Pérez-Payá E - BMC Struct. Biol. (2004)

Bottom Line: The determinants for fibril formation, as well as the possible physiological role are not fully understood.Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions.The putative influence of these properties with plant copper delivery will be addressed in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46100 Burjassot, València, Spain. helena.mira@uv.es

ABSTRACT

Background: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH.

Results: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide.

Conclusion: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

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Evidence for amyloid-like fibrils formed by peptide-1. (A). Visible spectra of the Congo red dye at two different pH values (circles pH 4.2 and squares pH 7.2) in the absence (empty symbols) and in the presence (filled symbols) of 50 μM of peptide-1. (B). Thioflavin T (10 μM) emission fluorescence spectra in Tris-HCl 20 mM pH 7.2 buffer in the absence (empty squares) and in the presence of peptide-1 at 200 μM (filled squares).
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Figure 6: Evidence for amyloid-like fibrils formed by peptide-1. (A). Visible spectra of the Congo red dye at two different pH values (circles pH 4.2 and squares pH 7.2) in the absence (empty symbols) and in the presence (filled symbols) of 50 μM of peptide-1. (B). Thioflavin T (10 μM) emission fluorescence spectra in Tris-HCl 20 mM pH 7.2 buffer in the absence (empty squares) and in the presence of peptide-1 at 200 μM (filled squares).

Mentions: Spectroscopic binding assays of peptide-1 at 50 μM to the diazo dye Congo red at two different pH values (Fig. 6A) show the typical red shift in wavelength and increase in intensity characteristic of amyloid-like fibrils [32]. The dye tioflavin T (ThT) is also used for the detection of amyloid fibrils [33]. Incubation of a peptide-1 solution at pH 7.5 produces an increase of ThT fluorescence (Fig. 6B). The oligomeric state of peptide-1 in solution was determined by analytical ultracentrifugation. At 10°C the sedimentation analysis (not shown) allowed the determinations of the Svedverg coefficients for peptide-1 (50 μM) at pH 4.1 and pH 8, which are compatible with molecular weights between 300 and 1000 kDa. These results suggest that peptide-1, at neutral and acidic pH values, has a high tendency to form high molecular weight aggregates that have amyloid-like fibril characteristics.


Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

Mira H, Vilar M, Esteve V, Martinell M, Kogan MJ, Giralt E, Salom D, Mingarro I, Peñarrubia L, Pérez-Payá E - BMC Struct. Biol. (2004)

Evidence for amyloid-like fibrils formed by peptide-1. (A). Visible spectra of the Congo red dye at two different pH values (circles pH 4.2 and squares pH 7.2) in the absence (empty symbols) and in the presence (filled symbols) of 50 μM of peptide-1. (B). Thioflavin T (10 μM) emission fluorescence spectra in Tris-HCl 20 mM pH 7.2 buffer in the absence (empty squares) and in the presence of peptide-1 at 200 μM (filled squares).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC425589&req=5

Figure 6: Evidence for amyloid-like fibrils formed by peptide-1. (A). Visible spectra of the Congo red dye at two different pH values (circles pH 4.2 and squares pH 7.2) in the absence (empty symbols) and in the presence (filled symbols) of 50 μM of peptide-1. (B). Thioflavin T (10 μM) emission fluorescence spectra in Tris-HCl 20 mM pH 7.2 buffer in the absence (empty squares) and in the presence of peptide-1 at 200 μM (filled squares).
Mentions: Spectroscopic binding assays of peptide-1 at 50 μM to the diazo dye Congo red at two different pH values (Fig. 6A) show the typical red shift in wavelength and increase in intensity characteristic of amyloid-like fibrils [32]. The dye tioflavin T (ThT) is also used for the detection of amyloid fibrils [33]. Incubation of a peptide-1 solution at pH 7.5 produces an increase of ThT fluorescence (Fig. 6B). The oligomeric state of peptide-1 in solution was determined by analytical ultracentrifugation. At 10°C the sedimentation analysis (not shown) allowed the determinations of the Svedverg coefficients for peptide-1 (50 μM) at pH 4.1 and pH 8, which are compatible with molecular weights between 300 and 1000 kDa. These results suggest that peptide-1, at neutral and acidic pH values, has a high tendency to form high molecular weight aggregates that have amyloid-like fibril characteristics.

Bottom Line: The determinants for fibril formation, as well as the possible physiological role are not fully understood.Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions.The putative influence of these properties with plant copper delivery will be addressed in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46100 Burjassot, València, Spain. helena.mira@uv.es

ABSTRACT

Background: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH.

Results: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide.

Conclusion: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

Show MeSH
Related in: MedlinePlus