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Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus : An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress

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ABSTRACT

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

No MeSH data available.


Structural composition vs. TFE concentration in rAcLEA solution as calculated from respective CD data using CDNN software.
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Figure 5: Structural composition vs. TFE concentration in rAcLEA solution as calculated from respective CD data using CDNN software.

Mentions: It is well established that TFE can induce α-helix folding in peptides (Buck, 1998; Boswell et al., 2014), as well as in unstructured proteins with a predisposition to form secondary structure such as LEA proteins (Shih et al., 2004; Rivera-Najera et al., 2014). Therefore the effect of TFE was evaluated on the rAcLEA conformation. Far UV CD spectra clearly show the tendency of rAcLEA to adopt helical structure as TFE concentration increases (Figure 4C). At TFE concentrations higher than 25%, the CD spectra of rAcLEA show the distinctive minima at 208 and 222 nm characteristic of α-helix structures (Muller et al., 2008). As TFE concentration increased up to 66% a gain of helical structure up 70.7% and a decreased in all the other types of secondary structure were observed (Figure 5 and Supplementary Table S1), this result being quantitatively confirmed using the CDNN program (Supplementary Table S1). In order to determine if this increase in helical content was accompanied with the formation of a structured core, the effect of temperature on rAcLEA dissolved in 50% TFE was assayed. It was found that the ellipticity signal at 208 and 222 nm was lost in a non-cooperative way (Figure 4D) and changes in CD signal were fully reversible at 25 and 50% TFE (Figure 4E). This strongly suggests that the helical segments induced by the addition of TFE are not arranged in a well-folded tertiary structure. To further explore the formation of tertiary structure, the CD spectra of rAcLEA in the aromatic region were also determined. In the absence of TFE, rAcLEA showed a weak signal in the region corresponding to Tyr and Phe residues, the intensity at 270 nm band being further decreased in the presence of TFE (Figure 4F), thus confirming the absence of TFE-induced tertiary structure formation.


Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus : An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress
Structural composition vs. TFE concentration in rAcLEA solution as calculated from respective CD data using CDNN software.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384071&req=5

Figure 5: Structural composition vs. TFE concentration in rAcLEA solution as calculated from respective CD data using CDNN software.
Mentions: It is well established that TFE can induce α-helix folding in peptides (Buck, 1998; Boswell et al., 2014), as well as in unstructured proteins with a predisposition to form secondary structure such as LEA proteins (Shih et al., 2004; Rivera-Najera et al., 2014). Therefore the effect of TFE was evaluated on the rAcLEA conformation. Far UV CD spectra clearly show the tendency of rAcLEA to adopt helical structure as TFE concentration increases (Figure 4C). At TFE concentrations higher than 25%, the CD spectra of rAcLEA show the distinctive minima at 208 and 222 nm characteristic of α-helix structures (Muller et al., 2008). As TFE concentration increased up to 66% a gain of helical structure up 70.7% and a decreased in all the other types of secondary structure were observed (Figure 5 and Supplementary Table S1), this result being quantitatively confirmed using the CDNN program (Supplementary Table S1). In order to determine if this increase in helical content was accompanied with the formation of a structured core, the effect of temperature on rAcLEA dissolved in 50% TFE was assayed. It was found that the ellipticity signal at 208 and 222 nm was lost in a non-cooperative way (Figure 4D) and changes in CD signal were fully reversible at 25 and 50% TFE (Figure 4E). This strongly suggests that the helical segments induced by the addition of TFE are not arranged in a well-folded tertiary structure. To further explore the formation of tertiary structure, the CD spectra of rAcLEA in the aromatic region were also determined. In the absence of TFE, rAcLEA showed a weak signal in the region corresponding to Tyr and Phe residues, the intensity at 270 nm band being further decreased in the presence of TFE (Figure 4F), thus confirming the absence of TFE-induced tertiary structure formation.

View Article: PubMed Central - PubMed

ABSTRACT

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

No MeSH data available.