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Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine.

Fremaux I, Mazères S, Brisson-Lougarre A, Arnaud M, Ladurantie C, Fournier D - BMC Biochem. (2002)

Bottom Line: The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature.It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologique, UMR 5068, Université Paul Sabatier, 31062, Toulouse, France. isa10yann@yahoo.fr

ABSTRACT

Background: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.

Results: The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.

Conclusion: It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.

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Orientation of cysteine 290 towards the center of the protein.
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Figure 2: Orientation of cysteine 290 towards the center of the protein.

Mentions: There are eight cysteines in the wild type cholinesterase [17], six are involved in intrachain disulfide bonds, one is involved in an interchain disulfide bond and one, at position 290 (328 with the precursor numbering) remains free [18]. This free cysteine is half buried in the protein, located in a loop near the disulfide bond formed by cysteines 292 and 307 (Fig. 1 and 2) [19]. When comparing the primary sequence of cholinesterases, it appears that this cysteine is not conserved and thus is not essential for the function of the enzyme. In the present study, we used mutagenesis to change this free cysteine for a hydrophobic residue with a similar size, a valine to enhance the stability of the enzyme.


Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine.

Fremaux I, Mazères S, Brisson-Lougarre A, Arnaud M, Ladurantie C, Fournier D - BMC Biochem. (2002)

Orientation of cysteine 290 towards the center of the protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117796&req=5

Figure 2: Orientation of cysteine 290 towards the center of the protein.
Mentions: There are eight cysteines in the wild type cholinesterase [17], six are involved in intrachain disulfide bonds, one is involved in an interchain disulfide bond and one, at position 290 (328 with the precursor numbering) remains free [18]. This free cysteine is half buried in the protein, located in a loop near the disulfide bond formed by cysteines 292 and 307 (Fig. 1 and 2) [19]. When comparing the primary sequence of cholinesterases, it appears that this cysteine is not conserved and thus is not essential for the function of the enzyme. In the present study, we used mutagenesis to change this free cysteine for a hydrophobic residue with a similar size, a valine to enhance the stability of the enzyme.

Bottom Line: The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature.It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologique, UMR 5068, Université Paul Sabatier, 31062, Toulouse, France. isa10yann@yahoo.fr

ABSTRACT

Background: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.

Results: The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.

Conclusion: It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.

Show MeSH