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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.

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NMR.1H-NMR spectrum of HisAcLEA purified from native conditions. Narrow signal distribution in amide region, between 6.5 and 8.5 ppm, strongly suggest the lack of a well-defined tridimentional structure distintive of intrinsically disordered proteins.
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Figure 3: NMR.1H-NMR spectrum of HisAcLEA purified from native conditions. Narrow signal distribution in amide region, between 6.5 and 8.5 ppm, strongly suggest the lack of a well-defined tridimentional structure distintive of intrinsically disordered proteins.

Mentions: rAcLEA obtained under native conditions was used to evaluate the structural conformation of the recombinant protein by proton nuclear magnetic resonance. Uni-dimensional 1H-NMR spectrum provides general overviews of protein structure because chemical shifts values are strongly related with the presence of different elements of secondary structure (Wishart et al., 1991; Mielke and Krishnan, 2009). Particularly, HN amide protons are widespread from 6 to 11 ppm in proteins with a well-defined tri-dimensional folding with a high content of α-helix and β-strand. In contrast, HN resonances of unfolded proteins with a random coil conformation collapse in a narrow region around 7–8 ppm (Singh et al., 2005). Figure 3 shows the 1H-NMR spectrum of native rAcLEA, as can be observed amide and aromatic protons are distributed between 6.8 and 8.6 ppm, suggesting a random conformation. Moreover, Hα resonances around 4.1 ppm have also a compact distribution, which is consistent with random coil as well the absence of splitting due to coupling in aliphatic signals in the 0.8–2.0 ppm range. This spectroscopic patterns indicate that methyl and methylene groups present in aliphatic amino acid lateral chains have free rotation without limitations due to steric impediment, suggesting that rAcLEA in the experimental conditions tested lacks secondary and tertiary structure. In fact, rAcLEA possesses the typical NMR profile for IDP previously observed in a LEA protein of T. aestivum (Sasaki et al., 2014) and a dehydrin of A. thaliana (Agoston et al., 2011).


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
NMR.1H-NMR spectrum of HisAcLEA purified from native conditions. Narrow signal distribution in amide region, between 6.5 and 8.5 ppm, strongly suggest the lack of a well-defined tridimentional structure distintive of intrinsically disordered proteins.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: NMR.1H-NMR spectrum of HisAcLEA purified from native conditions. Narrow signal distribution in amide region, between 6.5 and 8.5 ppm, strongly suggest the lack of a well-defined tridimentional structure distintive of intrinsically disordered proteins.
Mentions: rAcLEA obtained under native conditions was used to evaluate the structural conformation of the recombinant protein by proton nuclear magnetic resonance. Uni-dimensional 1H-NMR spectrum provides general overviews of protein structure because chemical shifts values are strongly related with the presence of different elements of secondary structure (Wishart et al., 1991; Mielke and Krishnan, 2009). Particularly, HN amide protons are widespread from 6 to 11 ppm in proteins with a well-defined tri-dimensional folding with a high content of α-helix and β-strand. In contrast, HN resonances of unfolded proteins with a random coil conformation collapse in a narrow region around 7–8 ppm (Singh et al., 2005). Figure 3 shows the 1H-NMR spectrum of native rAcLEA, as can be observed amide and aromatic protons are distributed between 6.8 and 8.6 ppm, suggesting a random conformation. Moreover, Hα resonances around 4.1 ppm have also a compact distribution, which is consistent with random coil as well the absence of splitting due to coupling in aliphatic signals in the 0.8–2.0 ppm range. This spectroscopic patterns indicate that methyl and methylene groups present in aliphatic amino acid lateral chains have free rotation without limitations due to steric impediment, suggesting that rAcLEA in the experimental conditions tested lacks secondary and tertiary structure. In fact, rAcLEA possesses the typical NMR profile for IDP previously observed in a LEA protein of T. aestivum (Sasaki et al., 2014) and a dehydrin of A. thaliana (Agoston et al., 2011).

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.


Related in: MedlinePlus