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Interactions of archaeal chromatin proteins Alba1 and Alba2 with nucleic acids.

Črnigoj M, Podlesek Z, Zorko M, Jerala R, Anderluh G, Ulrih NP - PLoS ONE (2013)

Bottom Line: Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT).The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures.Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.

View Article: PubMed Central - PubMed

Affiliation: Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

ABSTRACT

Background: Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823).

Methodology/principal findings: THESE TWO PEPTIDES WERE EXPRESSED AND THEIR INTERACTIONS WITH VARIOUS DNAS WERE STUDIED USING A COMBINATION OF VARIOUS EXPERIMENTAL TECHNIQUES: surface plasmon resonance, UV spectrophotometry, circular dichroism-spectropolarimetry, gel-shift assays, and isothermal titration calorimetry.

Conclusions/significance: Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.

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Native and SDS PAGE electrophoresis.Left: Native protein PAGE electrophoresis with 20% homogeneous gel for the Phast system. á, equinatoxin II; b´, Alba1; c´, Alba2; and d´, equimolar ratio of Alba1/Alba2. Right: SDS PAGE electrophoresis shows influence of disulphide bridges (±DTT) on dimerisation of Alba proteins. a, h, PageRuler protein ladder; b, e, Alba1; c, f, Alba2; d, g, equimolar ratio of Alba1/Alba2.
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pone-0058237-g002: Native and SDS PAGE electrophoresis.Left: Native protein PAGE electrophoresis with 20% homogeneous gel for the Phast system. á, equinatoxin II; b´, Alba1; c´, Alba2; and d´, equimolar ratio of Alba1/Alba2. Right: SDS PAGE electrophoresis shows influence of disulphide bridges (±DTT) on dimerisation of Alba proteins. a, h, PageRuler protein ladder; b, e, Alba1; c, f, Alba2; d, g, equimolar ratio of Alba1/Alba2.

Mentions: There are two known DNA sequences for the Alba proteins in the Archaea A. pernix: APE1832.1 (Alba1; Ape10b1) and APE1823 (Alba2; Ape10b2). Alba1 is composed of 94 amino-acid residues (10,336 Da) and has a theoretical isoelectric point (pI) of 9.5. Alba2 has 102 amino-acid residues (11,380 Da) and a theoretical pI of 9.1 [4], [8]. Both of the Alba proteins bound to the DNA in EMSAs regardless to the presence of a His-tag (removed via a thrombin cleavage site) (Figure 1). Similarly, no differences in DNA shift were observed if the Alba protein/DNA mixtures were incubated at higher temperatures (50°C) prior to EMSA. In the native protein acrylamide gel electrophoresis, the Alba proteins co-migrated with equinatoxin II (19.82 kDa, pI 10.2), which was used as a reference. Under the applied conditions, Alba1 appeared as a single band in the gel, while Alba2 and the equimolar mixture of both of these Alba proteins showed two bands, where the weaker band is likely to correspond to a monomer, and the stronger band to a dimer (Figure 2, left side).


Interactions of archaeal chromatin proteins Alba1 and Alba2 with nucleic acids.

Črnigoj M, Podlesek Z, Zorko M, Jerala R, Anderluh G, Ulrih NP - PLoS ONE (2013)

Native and SDS PAGE electrophoresis.Left: Native protein PAGE electrophoresis with 20% homogeneous gel for the Phast system. á, equinatoxin II; b´, Alba1; c´, Alba2; and d´, equimolar ratio of Alba1/Alba2. Right: SDS PAGE electrophoresis shows influence of disulphide bridges (±DTT) on dimerisation of Alba proteins. a, h, PageRuler protein ladder; b, e, Alba1; c, f, Alba2; d, g, equimolar ratio of Alba1/Alba2.
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pone-0058237-g002: Native and SDS PAGE electrophoresis.Left: Native protein PAGE electrophoresis with 20% homogeneous gel for the Phast system. á, equinatoxin II; b´, Alba1; c´, Alba2; and d´, equimolar ratio of Alba1/Alba2. Right: SDS PAGE electrophoresis shows influence of disulphide bridges (±DTT) on dimerisation of Alba proteins. a, h, PageRuler protein ladder; b, e, Alba1; c, f, Alba2; d, g, equimolar ratio of Alba1/Alba2.
Mentions: There are two known DNA sequences for the Alba proteins in the Archaea A. pernix: APE1832.1 (Alba1; Ape10b1) and APE1823 (Alba2; Ape10b2). Alba1 is composed of 94 amino-acid residues (10,336 Da) and has a theoretical isoelectric point (pI) of 9.5. Alba2 has 102 amino-acid residues (11,380 Da) and a theoretical pI of 9.1 [4], [8]. Both of the Alba proteins bound to the DNA in EMSAs regardless to the presence of a His-tag (removed via a thrombin cleavage site) (Figure 1). Similarly, no differences in DNA shift were observed if the Alba protein/DNA mixtures were incubated at higher temperatures (50°C) prior to EMSA. In the native protein acrylamide gel electrophoresis, the Alba proteins co-migrated with equinatoxin II (19.82 kDa, pI 10.2), which was used as a reference. Under the applied conditions, Alba1 appeared as a single band in the gel, while Alba2 and the equimolar mixture of both of these Alba proteins showed two bands, where the weaker band is likely to correspond to a monomer, and the stronger band to a dimer (Figure 2, left side).

Bottom Line: Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT).The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures.Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.

View Article: PubMed Central - PubMed

Affiliation: Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

ABSTRACT

Background: Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823).

Methodology/principal findings: THESE TWO PEPTIDES WERE EXPRESSED AND THEIR INTERACTIONS WITH VARIOUS DNAS WERE STUDIED USING A COMBINATION OF VARIOUS EXPERIMENTAL TECHNIQUES: surface plasmon resonance, UV spectrophotometry, circular dichroism-spectropolarimetry, gel-shift assays, and isothermal titration calorimetry.

Conclusions/significance: Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.

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