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Dissimilar roles of the four conserved acidic residues in the thermal stability of poly(A)-specific ribonuclease.

He GJ, Liu WF, Yan YB - Int J Mol Sci (2011)

Bottom Line: It was found that Mg(2+) significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner.All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg(2+)-dependent behavior.The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China; E-Mails: he-gj06@mails.tsinghua.edu.cn (G.-J.H.); liuwf@mail.tsinghua.edu.cn (W.-F.L.).

ABSTRACT
Divalent metal ions are essential for the efficient catalysis and structural stability of many nucleotidyl-transfer enzymes. Poly(A)-specific ribonuclease (PARN) belongs to the DEDD superfamily of 3'-exonucleases, and the active site of PARN contains four conserved acidic amino acid residues that coordinate two Mg(2+) ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg(2+) significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner. All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg(2+)-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg(2+) ions, while metal B might be more important in PARN structural stability. The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule.

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Effects of mutations on PARN secondary and tertiary structures monitored by far-UV CD (A), intrinsic Trp fluorescence (B) and ANS fluorescence (C). The proteins were dissolved in buffer T containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM DTT, 0.2 mM EDTA and 20% (v/v) glycerol in the presence or absence of 3 mM MgCl2. The final protein concentration was 0.2 mg/mL. The excitation wavelength of the intrinsic fluorescence was 295 nm, while that of the ANS fluorescence was 380 nm. The resultant spectra were obtained by the subtraction of the spectra of the buffer. All spectroscopic experiments were carried out at 25 °C.
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f2-ijms-12-02901: Effects of mutations on PARN secondary and tertiary structures monitored by far-UV CD (A), intrinsic Trp fluorescence (B) and ANS fluorescence (C). The proteins were dissolved in buffer T containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM DTT, 0.2 mM EDTA and 20% (v/v) glycerol in the presence or absence of 3 mM MgCl2. The final protein concentration was 0.2 mg/mL. The excitation wavelength of the intrinsic fluorescence was 295 nm, while that of the ANS fluorescence was 380 nm. The resultant spectra were obtained by the subtraction of the spectra of the buffer. All spectroscopic experiments were carried out at 25 °C.

Mentions: Spectroscopic experiments were carried out to assess the effect of mutation on PARN structure. The circular dichroism (CD) data shown in Figure 2A indicated that the effects of the mutations on the secondary structure contents of PARN were minor. The four mutations affect PARN secondary structures dissimilarly: No significant changes were observed for the D28A and D292A mutations, while there was an ∼10% increase in the absolute value of ellipticity at 222 nm ([θ222]) for the E30A and D382A mutations. With the addition of 3 mM Mg2+, a minor increase of [θ222] was observed for the WT protein. As for the mutants, the [θ222] value changed little for D28A; there was a slight decrease for D382A and a significant decrease to the value close to that of the WT protein for E30A. However an ∼15% increase was found for D292A. These results indicated that the mutations either slightly increased or did not affect the percentages of the regular secondary structure contents of PARN. The coordination of Mg2+ could rescue the disturbance in secondary structures induced by most mutations except D292A.


Dissimilar roles of the four conserved acidic residues in the thermal stability of poly(A)-specific ribonuclease.

He GJ, Liu WF, Yan YB - Int J Mol Sci (2011)

Effects of mutations on PARN secondary and tertiary structures monitored by far-UV CD (A), intrinsic Trp fluorescence (B) and ANS fluorescence (C). The proteins were dissolved in buffer T containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM DTT, 0.2 mM EDTA and 20% (v/v) glycerol in the presence or absence of 3 mM MgCl2. The final protein concentration was 0.2 mg/mL. The excitation wavelength of the intrinsic fluorescence was 295 nm, while that of the ANS fluorescence was 380 nm. The resultant spectra were obtained by the subtraction of the spectra of the buffer. All spectroscopic experiments were carried out at 25 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116163&req=5

f2-ijms-12-02901: Effects of mutations on PARN secondary and tertiary structures monitored by far-UV CD (A), intrinsic Trp fluorescence (B) and ANS fluorescence (C). The proteins were dissolved in buffer T containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM DTT, 0.2 mM EDTA and 20% (v/v) glycerol in the presence or absence of 3 mM MgCl2. The final protein concentration was 0.2 mg/mL. The excitation wavelength of the intrinsic fluorescence was 295 nm, while that of the ANS fluorescence was 380 nm. The resultant spectra were obtained by the subtraction of the spectra of the buffer. All spectroscopic experiments were carried out at 25 °C.
Mentions: Spectroscopic experiments were carried out to assess the effect of mutation on PARN structure. The circular dichroism (CD) data shown in Figure 2A indicated that the effects of the mutations on the secondary structure contents of PARN were minor. The four mutations affect PARN secondary structures dissimilarly: No significant changes were observed for the D28A and D292A mutations, while there was an ∼10% increase in the absolute value of ellipticity at 222 nm ([θ222]) for the E30A and D382A mutations. With the addition of 3 mM Mg2+, a minor increase of [θ222] was observed for the WT protein. As for the mutants, the [θ222] value changed little for D28A; there was a slight decrease for D382A and a significant decrease to the value close to that of the WT protein for E30A. However an ∼15% increase was found for D292A. These results indicated that the mutations either slightly increased or did not affect the percentages of the regular secondary structure contents of PARN. The coordination of Mg2+ could rescue the disturbance in secondary structures induced by most mutations except D292A.

Bottom Line: It was found that Mg(2+) significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner.All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg(2+)-dependent behavior.The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China; E-Mails: he-gj06@mails.tsinghua.edu.cn (G.-J.H.); liuwf@mail.tsinghua.edu.cn (W.-F.L.).

ABSTRACT
Divalent metal ions are essential for the efficient catalysis and structural stability of many nucleotidyl-transfer enzymes. Poly(A)-specific ribonuclease (PARN) belongs to the DEDD superfamily of 3'-exonucleases, and the active site of PARN contains four conserved acidic amino acid residues that coordinate two Mg(2+) ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg(2+) significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner. All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg(2+)-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg(2+) ions, while metal B might be more important in PARN structural stability. The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule.

Show MeSH
Related in: MedlinePlus