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.

Modular strategy for the semisynthesis of a K+ channel: investigating interactions of the pore helix.

Chemical synthesis is a powerful method for precise modification of the structural and electronic properties of proteins. The difficulties in the synthesis and purification of peptides containing transmembrane segments have presented obstacles to the chemical synthesis of integral membrane proteins. Here, we present a modular strategy for the semisynthesis of integral membrane proteins in which solid-phase peptide synthesis is limited to the region of interest, while the rest of the protein is obtained by recombinant means. This modular strategy considerably simplifies the synthesis and purification steps that have previously hindered the chemical synthesis of integral membrane proteins. We develop a SUMO fusion and proteolysis approach for obtaining the N-terminal cysteine containing membrane-spanning peptides required for the semisynthesis. We demonstrate the feasibility of the modular approach by the semisynthesis of full-length KcsA K(+) channels in which only regions of interest, such as the selectivity filter or the pore helix, are obtained by chemical synthesis. The modular approach is used to investigate the hydrogen bond interactions of a tryptophan residue in the pore helix, tryptophan 68, by substituting it with the isosteric analogue, beta-(3-benzothienyl)-l-alanine (3BT). A functional analysis of the 3BT mutant channels indicates that the K(+) conduction and selectivity of the 3BT mutant channels are similar to those of the wild type, but the mutant channels show a 3-fold increase in Rb(+) conduction. These results suggest that the hydrogen bond interactions of tryptophan 68 are essential for optimizing the selectivity filter for K(+) conduction over Rb(+) conduction.

Semisynthesis of K+ channels.

The ability to selectively conduct K(+) ions is central to the function of K(+) channels. Selection for K(+) and rejection of Na(+) takes place in a conserved structural element referred to as the selectivity filter. The selectivity filter consists of four K(+)-specific ion binding sites that are created using predominantly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, experimental manipulation of the ion binding sites in the selectivity filter is not possible using traditional site directed mutagenesis. The limited suitability of the site-directed mutagenesis for studies on the selectivity filter has motivated the development of a semisynthesis approach, which enables the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that are presently used in our laboratory for the semisynthesis of the bacterial K(+) channel, KcsA. We show the introduction of a spectroscopic probe into the KcsA channel using semisynthesis. We also review previous applications of semisynthesis in investigations of K(+) channels. While the protocols described in this chapter are for the KcsA K(+) channel, we anticipate that similar protocols will also be applicable for the semisynthesis of other integral membrane proteins.