Protein production and purification
cDNA encoding wild type drosophila AChE and mutant were expressed with the baculovirus system [29]. This enzyme is a dimer anchor to the membrane via a glycolipid [30]. We expressed a soluble dimeric form obtained by deletion of the hydrophobic peptide, precursor of the glycolipid anchor at the C-terminal end [31]. Furthermore, the enzyme was deleted of the loop from amino-acids 103 to 136 which has been replaced by 3 histidines. Secreted AChE was purified to homogeneity using the following steps: ammonium sulfate precipitation, ultrafiltration with a 10 kDa cutoff membrane, NTA-nickel chromatography, affinity chromatography with procainamide as ligand and gel filtration [6]. Activity was recorded at 25°C in 25 mM phosphate buffer pH 7, with 1 mM acetylthiocholine iodide as substrate using the method of Ellman et al. [32]. Mutations did not alter the specific activity of the proteins. Molecular modeling of mutated residues was performed from the structure solved by Harel et al. [[33], ref 1QO9] by homology using Swiss model server available at http://expasy.org/swissmod/. Residue numbering followed that of the mature protein.
Production
One liter containing 106 Sf9 cells was infected with more than 107 virus. After four days incubation at 28°C, the cells were lyzed by adding Triton X-100 (0.1%). Amount of AChE was estimated by active site titration using an irreversible inhibitor, chlorpyriphos oxon [34]. The mean of at least five independent productions was compared to the production of the wild type enzyme.
Denaturation
All denaturation experiments were performed with 10 picomoles of enzyme in one ml 25 mM phosphate buffer pH7 at 25°C. AChE was incubated in denaturing conditions. Aliquots were taken at regular intervals, diluted 20 x to stop the action of the denaturing agent and the remaining activity was measured.
To analyze thermosensitivity, enzymes were incubated at 50°C with 1 mg/ml Bovine Serum Albumin in the buffer. Before recording the remaining activity, aliquots were mixed with cold buffer chilled on ice and the solution was incubated at 25°C for ten minutes to eliminate the reversible component [35]. For urea denaturation, unfolding of AChE was induced by adding 4 M urea into the incubation buffer. The effect of organic solvent was followed by incubation of the enzyme in 20% acetonitrile. The effect of protease sensitivity was determined by incubation of AChE with 0.1 mg/ml pronase.
Three to nine batches of each mutant were produced and purified. Three repeats were performed for each batch and each denaturing agent. Significance of difference observed in stability was tested using the Mann Whitney test.
Coulombic interaction estimation
In order to estimate the effects of charge distribution on each of the mutants, the contributions from electrostatic interactions were extracted from the total energy of the minimized structures of the molecules. The minimization was performed with the GROMACS software [36], using the simulated annealing protocol. Position restraints were imposed on the whole molecule with the exception of the mutated residue, which was allowed to adopt the energetically favorable configuration. The Coulomb's interactions were normalized by subtracting the value obtained for the wild type protein and by rescaling the numbers by 80, to take into account the dielectric constant of water.
Answers to all your Biology Questions
