Work

Semisynthesis of NaK, a Na+ and K+ Conducting Ion Channel

Year: 2010

Type: article

Abstract: 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.

Source: Biochemistry

DOI: 10.1021/bi100413z

Authors: Kellie M. Linn, Mehabaw G. Derebe, Youxing Jiang, Francis I. Valiyaveetil

Institutions: Oregon Health & Science University, The University of Texas Southwestern Medical Center

Language: English

Topic: Ion channel regulation and function

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: Howard Hughes Medical Institute, National Institute of General Medical Sciences

Awards: G2796398753, Probing functional mechanisms in K+ channels using unnatural mutagenesis, Structural mechanisms of ion selectivity and gating in tetrameric cation channels, G7229826939