Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine

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 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.

Insecticide Detection through Protein Engineering of Nippostrongylus brasiliensis Acetylcholinesterase B

“…The reason for this activity loss is seen in the humidity of the enzyme layer when stored at room temperature overnight. The negative effect of moisture on the stability of AChE was already reported elsewhere. , We also tested the reactivation of the biosensor, a further criterion for its functionality. The AChE activity of the new and 17-month-old biosensors could be completely reactivated after each measurement using 2-PAM.…”

“…In the case of Torpedo marmorata 23 and D. melanogaster AChE, problems in enzyme stability were reported. Several approaches were performed to increase the stability of AChE, for example, by adding protective compounds or, in the case of the D melanogaster AChE, by protein engineering . The exchange of a cysteine residue, which is not involved in an intra- or intermolecular disulfide bridge, by valine increased the temperature stability of the D. melanogaster AChE.…”

The Interaction between Thermodynamic Stability and Buried Free Cysteines in Regulating the Functional Half-Life of Fibroblast Growth Factor-1

“…Due to the negative consequences on protein structure caused by thiol adduct formation of buried free cysteines, mutational substitution of such residues is often accompanied by a notable increase in functional half-life. [9][10][11][12][13] An analysis of a set of 131 nonhomologous singledomain protein X-ray structures (1.95 Å resolution or better) by Petersen et al reported that the prevalence of free-cysteine residues in proteins is 0.5% (or, typically, one free cysteine in an average size protein); furthermore, 50% of these free cysteines are buried within the protein interior. 14 Thus, although potentially highly problematic for protein therapeutic application, the presence of buried free cysteines in proteins is a surprisingly common occurrence; some familiar examples include fibroblast growth factors (FGFs), interleukin-2, β-interferon, granulocyte colony-stimulating factor, and insulin-like growth factor-binding protein-1 (with the majority of these being approved human therapeutics).…”

Thermostability Improvement of L-Asparaginase from Acinetobacter soli via Consensus-Designed Cysteine Residue Substitution

“…However, the –SH groups of free cysteine residues can easily be oxidized by metal ions such as Cu 2+ , which negatively affects protein folding [ 22 ]. The introduction of a suitable couple of cysteine residues to form a stable covalent bond and the substitution of a cysteine residue with different amino acids was proven effective in eliminating the negative effect of free –SH groups [ 23 , 24 ]. There are many successful cases of cysteine residues substituted by alanine, serine, and valine for increasing the thermostability of enzymes [ 25 , 26 ].…”