Semisynthesis of NaK, a Na⁺ and K⁺ conducting ion channel

In this contribution, we describe the semisynthesis of NaK, a bacterial nonselective cation channel. In the semisynthesis, the NaK polypeptide is assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction. We describe a temporary tagging strategy for the purification of the hydrophobic synthetic peptide and demonstrate the efficient ligation of the synthetic peptide with the recombinant peptide thioester to form the semisynthetic NaK polypeptide. Following assembly, the NaK polypeptide is folded in vitro to the native state using lipid vesicles. Functional characterization of the folded semisynthetic NaK channels indicates that it is functionally similar to the wild-type protein. We used semisynthesis to substitute aspartate 66 in the selectivity filter region of the NaK channel with the unnatural amino acids homoserine and cysteine sulfonic acid. Functional analysis of these mutants suggests that the presence of a negatively charged residue in the vicinity of the ion binding sites is necessary for optimal flux of ions through the NaK channel.