Background: Insecticide resistance is now common in insects due to the frequent use of chemicals to control them, which provides a useful tool to study the adaptation of eukaryotic genome to new environments. Although numerous potential mutations may provide high level of resistance, only few alleles are found in insect natural populations. Then, we hypothesized that only alleles linked to the highest fitness in the absence of insecticide are selected.
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 8
th
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.
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 8 th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.
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