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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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Conformational characterisation of peptide-1. (A). Tris-Tricine electrophoretic mobility of peptide-1 and peptide-122. (see Table 1 for theoretical molecular weights). Standard molecular weight markers are shown on the left. (B). far-UV CD spectra as a function of pH at 10°C. The concentration of peptide-1 was 50 μM. pH values are indicated in the spectra. The insert shows the dependence with the temperature of the molar ellipticity at 198 nm from spectra acquired at pH 6.8. (C). 1H NMR monodimensional spectra of peptide-1 (50 μM) in water at different pH values.
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Figure 4: Conformational characterisation of peptide-1. (A). Tris-Tricine electrophoretic mobility of peptide-1 and peptide-122. (see Table 1 for theoretical molecular weights). Standard molecular weight markers are shown on the left. (B). far-UV CD spectra as a function of pH at 10°C. The concentration of peptide-1 was 50 μM. pH values are indicated in the spectra. The insert shows the dependence with the temperature of the molar ellipticity at 198 nm from spectra acquired at pH 6.8. (C). 1H NMR monodimensional spectra of peptide-1 (50 μM) in water at different pH values.

Mentions: Peptide-1 (MWteo = 2267 Da) was analyzed by means of Tris-tricine electrophoresis and, as the C-domain did, it showed an altered electrophoretic mobility and an apparent molecular weight close to 26.6 kDa (Fig. 4A). Insertion of two additional alanines in the middle of the peptide-1 sequence generates a peptide (peptide-122 – Table 1) that when plotted in an α-helical conformation would lost the observed charge segregation for the peptide-1 (Fig. 1C). However, peptide-122, would retain the capability to adopt an amphipathic β-sheet conformation. The altered electrophoretic mobility obtained for peptide-122 (Fig. 4A) suggests that the oligomerization observed for these peptides is more related to the adoption of an amphipathic β-sheet conformation than to the adoption of an α-helical conformation with charge segregation. Furthermore, shortened synthetic peptide analogues from peptide-1 with amino acid deletions of two (peptide-118), six (peptide-114) and ten (peptide-110) from the N-terminal (Table 1) were also analysed in order to define the minimum peptide length able to drive oligomerization. Peptide-118 and peptide-114, but not peptide-110, showed altered electrophoretic mobility (data not shown).


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)

Conformational characterisation of peptide-1. (A). Tris-Tricine electrophoretic mobility of peptide-1 and peptide-122. (see Table 1 for theoretical molecular weights). Standard molecular weight markers are shown on the left. (B). far-UV CD spectra as a function of pH at 10°C. The concentration of peptide-1 was 50 μM. pH values are indicated in the spectra. The insert shows the dependence with the temperature of the molar ellipticity at 198 nm from spectra acquired at pH 6.8. (C). 1H NMR monodimensional spectra of peptide-1 (50 μM) in water at different pH values.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Conformational characterisation of peptide-1. (A). Tris-Tricine electrophoretic mobility of peptide-1 and peptide-122. (see Table 1 for theoretical molecular weights). Standard molecular weight markers are shown on the left. (B). far-UV CD spectra as a function of pH at 10°C. The concentration of peptide-1 was 50 μM. pH values are indicated in the spectra. The insert shows the dependence with the temperature of the molar ellipticity at 198 nm from spectra acquired at pH 6.8. (C). 1H NMR monodimensional spectra of peptide-1 (50 μM) in water at different pH values.
Mentions: Peptide-1 (MWteo = 2267 Da) was analyzed by means of Tris-tricine electrophoresis and, as the C-domain did, it showed an altered electrophoretic mobility and an apparent molecular weight close to 26.6 kDa (Fig. 4A). Insertion of two additional alanines in the middle of the peptide-1 sequence generates a peptide (peptide-122 – Table 1) that when plotted in an α-helical conformation would lost the observed charge segregation for the peptide-1 (Fig. 1C). However, peptide-122, would retain the capability to adopt an amphipathic β-sheet conformation. The altered electrophoretic mobility obtained for peptide-122 (Fig. 4A) suggests that the oligomerization observed for these peptides is more related to the adoption of an amphipathic β-sheet conformation than to the adoption of an α-helical conformation with charge segregation. Furthermore, shortened synthetic peptide analogues from peptide-1 with amino acid deletions of two (peptide-118), six (peptide-114) and ten (peptide-110) from the N-terminal (Table 1) were also analysed in order to define the minimum peptide length able to drive oligomerization. Peptide-118 and peptide-114, but not peptide-110, showed altered electrophoretic mobility (data not shown).

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