Salty Enzymes: Protein Splicing Dependence on Salt Concentrations

Halophiles are organisms that thrive in very high salt concentrations. Enzymes from halophiles tend to have a negative surface charge and a low isoelectric point to allow for stable folding. We are interested in converting a halophilic enzyme that requires high salt for activity and proper folding into one that is functional at more moderate salt concentrations. Our model enzyme is DNA polymerase II from Halobacterium salinarum, which contains an intein. Inteins are intervening peptide sequences within proteins that are spliced out from their flanking polypeptides, or exteins, which then ligate to form a functional protein. We have induced mutations to the intein that facilitate a splicing reaction at lower salt concentrations. Changing negatively charged amino acids on the surface of the intein to corresponding neutral or positive charged residues observed in homologous inteins in non-halophilic extremophiles induces the splicing reaction at lower salt concentrations, especially when coupled with a change of the C-terminal Gln to the more highly conserved Asn. As few as two residue changes can dramatically alter the salt-dependence of the activity, showing that subtle changes to surface residues can result in conditional folding and activity.