Mutations to hydrophilic residue may disrupt the bond and thus decrease the probability of the protein reaching its active conformation. It seems that these two residues are engaged in a hydrophobic bond with Val 14 helping to position the loop from Ala 50 to Thr 60 in connection with the N-terminus peptide of the protein (Fig. The mutations V56R and L57R decreased production to 10%. This amino-acid is partly buried behind proline 443 and we can assume that its hydrophobicity is important for the folding of the protein. One mutation drastically decreased production: the mutation M194R decreased production to 0.5% i.e. 1), most probably reflecting their influence on folding efficiency. Several mutations affected the production of the protein (Fig. Production of the wild type was 60 nmoles per liter (5 mg/L). Hydrophobic residues were chosen by visual examination of the structure and arginine was chosen because the guanidinium group is the most polar of all the common amino-acid residues found in proteins.Д®ffect of mutations on protein production Here we tested this strategy by mutating several hydrophobic residues scattered at the surface of Drosophila AChE to arginine. Several examples showed that the change of hydrophobicity to hydrophilicity of amino-acid residues exposed to the solvent at the surface of proteins is an efficient stategy to stabilize proteins: i) Analysis of protein sequences showed a strong bias for hydrophilic residues and against large hydrophobic residues at most surface positions, ii) Mutagenesis showed that hydrophilic amino acids at surface positions is stabilizing Mutagenesis showed that hydrophilic amino acids at surface positions have stabilizing effect, while placing a hydrophobic residue in a solvent-exposed position causes destabilization, iii) The proportion of hydrophobic residues at the surface of proteins from hyperthermophilic species was found to be reduced compared to the proportion in their mesophilic counterparts, however, this observation is under debate. We thus hypothesized that decreasing the hydrophobicity at the surface of the protein may have some stabilizing effects. Addition of protein in the solution would compete with hydrophobic surfaces and protect the enzyme. This suggested that denaturation occurred by interaction of the hydrophobic region at the surface of AChE with tube walls or air-solvent interface. Irreversible denaturation of AChE at room temperature can be minimized by increasing the protein concentration in the sample, either by increasing the enzyme concentration or by addition of another protein such as BSA. This method could have the additional advantage of stabilizing the enzyme during its synthesis leading to higher production and higher purification yields. Another way to stabilize the enzyme is to use in vitro mutagenesis to modify the primary structure of the protein. Alternatively, stabilization may also be achieved by encapsulation in liposomes. It can be stabilized by adding some molecules in the solution such as reversible inhibitors, polyethylene glycol or protein, provoking protein-protein interactions. But like most enzymes from mesophilic organisms, Drosophila AChE is not stable, and this instability precludes its utilization in biosensors. Drosophila AChE was found to be the most sensitive enzyme when compared to enzymes of non-insect origin and in-vitro-mutagenesis was used to select enzymes up to 300-fold more sensitive. This enzyme is irreversibly inhibited by organophosphate and carbamate pesticides leading to its use in biosensors to detect traces of these compounds in environment. Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine hydrolase, which catalyzes the hydrolysis of the neurotransmitter acetylcholine.

Charge on hydrophobic amino acids