A strategy for stabilization of CutA1 proteins due to ion-ion interactions at temperatures of over 100 °C.

In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 degrees C, we constructed many mutants of the CutA1 protein (EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 degrees C. The hydrophobic mutant of EcCutA1 (Ec0VV) with denaturation temperature (T-d) of 113.2 degrees C was used as a template for mutations. The highest T-d of Ec0VV mutants substituted by a single charged residue was 118.4 degrees C. Multiple ion mutants were also constructed by combination of single mutants and found to have an increased thermostability. The highest stability of multiple mutants was a mutant substituted by nine charged residues that had a Td of 142.2 degrees C. To evaluate the energy of ion-ion interactions of mutant proteins, we used the structural ensemble obtained by a molecular dynamics simulation at 300 K. The T-d of ionic mutants linearly increases with the increments of the computed energy of ion-ion interactions for ionic mutant proteins even up to the temperatures near 140 degrees C, suggesting that ion-ion interactions cumulatively contribute to the stabilization of a protein at high temperatures.