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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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TEM images for peptide-1. TEM images of the replicas obtained after freeze-fixation and freeze-drying of a 100 μM aqueous solution of the peptide-1 at different pHs.
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Figure 7: TEM images for peptide-1. TEM images of the replicas obtained after freeze-fixation and freeze-drying of a 100 μM aqueous solution of the peptide-1 at different pHs.

Mentions: In order to further characterise the aggregation process of peptide-1 in solution, eight different preparations of peptide solutions were freeze-fixed and freeze-dried to preserve the aggregate structures in solution for subsequent transmission electron microscopy (TEM) imaging. Two solutions at 10 and 100 μM of peptide-1 were prepared and divided in four fractions, being each fraction adjusted to different pH values (3, 5, 7 and 9) with HCl or NaOH. Different kinds of aggregates were observed depending on pH of the solutions. The fibrils obtained at pH 5 and 7 revealed by TEM are long and unbranched with diameters of 8 nm and up to 1000 nm length (Fig. 7). This fibrillar material appears similar to the protofilaments observed in other amyloidogenic systems [34,35]. In contrast at pH 3 and 9 peptide-1 aggregates giving irregular structures (Fig. 7).


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)

TEM images for peptide-1. TEM images of the replicas obtained after freeze-fixation and freeze-drying of a 100 μM aqueous solution of the peptide-1 at different pHs.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: TEM images for peptide-1. TEM images of the replicas obtained after freeze-fixation and freeze-drying of a 100 μM aqueous solution of the peptide-1 at different pHs.
Mentions: In order to further characterise the aggregation process of peptide-1 in solution, eight different preparations of peptide solutions were freeze-fixed and freeze-dried to preserve the aggregate structures in solution for subsequent transmission electron microscopy (TEM) imaging. Two solutions at 10 and 100 μM of peptide-1 were prepared and divided in four fractions, being each fraction adjusted to different pH values (3, 5, 7 and 9) with HCl or NaOH. Different kinds of aggregates were observed depending on pH of the solutions. The fibrils obtained at pH 5 and 7 revealed by TEM are long and unbranched with diameters of 8 nm and up to 1000 nm length (Fig. 7). This fibrillar material appears similar to the protofilaments observed in other amyloidogenic systems [34,35]. In contrast at pH 3 and 9 peptide-1 aggregates giving irregular structures (Fig. 7).

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