RESUMO
BACKGROUND: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization. RESULTS: To create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cbeta of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50 degrees C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies. CONCLUSION: Addition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation.
Assuntos
Acetilcolinesterase/química , Cistina/química , Dissulfetos/química , Proteínas de Drosophila/química , Acetonitrilas/farmacologia , Acetilcolinesterase/genética , Acetiltiocolina/farmacologia , Animais , Baculoviridae , DNA Complementar/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Temperatura Alta , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pronase/farmacologia , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Solventes/farmacologia , Ureia/farmacologiaRESUMO
Xerocomus chrysenteron is an edible mushroom with insecticidal properties. In an earlier work, we found that proteins are responsible for this toxicity. Here we describe the purification of a approximately 15 kDa lectin, named XCL, from the mushroom. Its cDNA and gDNA were cloned by PCR strategies and a recombinant form was expressed in Escherichia coli. Sequence alignments and sugar specificity showed that this protein is the third member of a new saline-soluble lectin family present in fungi. This protein, either purified from mushroom or expressed in vitro in E. coli, was found to be toxic to some insects, such as the dipteran Drosophila melanogaster and the hemipteran, Acyrthosiphon pisum. The lectin possesses a high insecticidal activity compared to lectin isolated from leguminosae (Lathyrus ochrus) or from the snowdrop (Galanthus nivalis).
Assuntos
Basidiomycota , Proteínas Fúngicas/isolamento & purificação , Inseticidas , Lectinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Basidiomycota/química , Basidiomycota/genética , Basidiomycota/metabolismo , Clonagem Molecular , Drosophila melanogaster , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/toxicidade , Genes Fúngicos , Lectinas/química , Lectinas/genética , Lectinas/toxicidade , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Alinhamento de Sequência , Testes de ToxicidadeRESUMO
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.