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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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(A) Representative time-course aggregation during PARN heated at 55 °C in the presence of various amounts of Mg2+. The aggregation was monitored by the absorbance at 400 nm on an Ultraspec 4300 pro UV/Visible spectrophotometer. The final protein concentration was 0.1 or 0.2 mg/mL. The raw data, which are presented as open symbols, were recorded every 2 s. The fitted curves are drawn as solid lines. The concentrations of Mg2+ (0–6 mM) and PARN are labeled. (B–D) Mg2+ dependence of the aggregation kinetic parameters. The parameters were obtained by fitting the raw data with Equation (1). The errors of the fitted parameters were within 5%.
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f3-ijms-12-02901: (A) Representative time-course aggregation during PARN heated at 55 °C in the presence of various amounts of Mg2+. The aggregation was monitored by the absorbance at 400 nm on an Ultraspec 4300 pro UV/Visible spectrophotometer. The final protein concentration was 0.1 or 0.2 mg/mL. The raw data, which are presented as open symbols, were recorded every 2 s. The fitted curves are drawn as solid lines. The concentrations of Mg2+ (0–6 mM) and PARN are labeled. (B–D) Mg2+ dependence of the aggregation kinetic parameters. The parameters were obtained by fitting the raw data with Equation (1). The errors of the fitted parameters were within 5%.

Mentions: In a previous study, we found that 3 mM Mg2+ can protect PARN against thermal inactivation below 60 °C, but it accelerates protein thermal aggregation at 61 °C [12]. A similar result was observed when PARN was heated at 55 °C (Figure 3A), where PARN thermal aggregation revealed Mg2+- and protein-concentration dependent manner. The aggregation kinetics was analyzed by considering the protein aggregation process as an n-th order reaction, and the following equation could be used for the fitting of the data [21](1)A = Alim (1 − exp(−kn(t − t0)n)where t is the time of incubation at a given temperature, Alim is the A400 value at the infinite time and kn is the rate constant of the n-th order reaction. Under all conditions, the aggregation was found to be dominated by a first-order kinetics with n = 1.17 ± 0.08. To facilitate the comparison of the parameters, all the data were fitted by assuming n = 1. The results shown in Figure 3 indicated that all the three parameters (t0, k and Alim) were significantly affected by the addition of Mg2+ (F test, P < 0.0001). Unexpectedly, the three parameters revealed inconsistent results with the increase of [Mg2+]: The increase in t0 and the decrease in k implied that Mg2+ could inhibit the rate of PARN aggregation, whereas the increase in Alim suggested that Mg2+ increased the amounts or size of aggregates.


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)

(A) Representative time-course aggregation during PARN heated at 55 °C in the presence of various amounts of Mg2+. The aggregation was monitored by the absorbance at 400 nm on an Ultraspec 4300 pro UV/Visible spectrophotometer. The final protein concentration was 0.1 or 0.2 mg/mL. The raw data, which are presented as open symbols, were recorded every 2 s. The fitted curves are drawn as solid lines. The concentrations of Mg2+ (0–6 mM) and PARN are labeled. (B–D) Mg2+ dependence of the aggregation kinetic parameters. The parameters were obtained by fitting the raw data with Equation (1). The errors of the fitted parameters were within 5%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijms-12-02901: (A) Representative time-course aggregation during PARN heated at 55 °C in the presence of various amounts of Mg2+. The aggregation was monitored by the absorbance at 400 nm on an Ultraspec 4300 pro UV/Visible spectrophotometer. The final protein concentration was 0.1 or 0.2 mg/mL. The raw data, which are presented as open symbols, were recorded every 2 s. The fitted curves are drawn as solid lines. The concentrations of Mg2+ (0–6 mM) and PARN are labeled. (B–D) Mg2+ dependence of the aggregation kinetic parameters. The parameters were obtained by fitting the raw data with Equation (1). The errors of the fitted parameters were within 5%.
Mentions: In a previous study, we found that 3 mM Mg2+ can protect PARN against thermal inactivation below 60 °C, but it accelerates protein thermal aggregation at 61 °C [12]. A similar result was observed when PARN was heated at 55 °C (Figure 3A), where PARN thermal aggregation revealed Mg2+- and protein-concentration dependent manner. The aggregation kinetics was analyzed by considering the protein aggregation process as an n-th order reaction, and the following equation could be used for the fitting of the data [21](1)A = Alim (1 − exp(−kn(t − t0)n)where t is the time of incubation at a given temperature, Alim is the A400 value at the infinite time and kn is the rate constant of the n-th order reaction. Under all conditions, the aggregation was found to be dominated by a first-order kinetics with n = 1.17 ± 0.08. To facilitate the comparison of the parameters, all the data were fitted by assuming n = 1. The results shown in Figure 3 indicated that all the three parameters (t0, k and Alim) were significantly affected by the addition of Mg2+ (F test, P < 0.0001). Unexpectedly, the three parameters revealed inconsistent results with the increase of [Mg2+]: The increase in t0 and the decrease in k implied that Mg2+ could inhibit the rate of PARN aggregation, whereas the increase in Alim suggested that Mg2+ increased the amounts or size of aggregates.

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