Lipid molecules surround an ion channel in its native environment of cellular membranes. The importance of the lipid bilayer and the role of lipid protein interactions in ion channel structure and function are not well understood. Here we demonstrate that the bacterial potassium channel KcsA binds a negatively charged lipid molecule. We have defined the potential binding site of the lipid molecule on KcsA by X-ray crystallographic analysis of a complex of KcsA with a monoclonal antibody Fab fragment. We also demonstrate that lipids are required for the in vitro refolding of the KcsA tetramer from the unfolded monomeric state. The correct refolding of the KcsA tetramer requires lipids, but it is not dependent on negatively charged lipids as refolding takes place in the absence of such lipids. We confirm that the presence of negatively charged lipids is required for ion conduction through the KcsA potassium channel, suggesting that the lipid bound to KcsA is important for ion channel function.

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose 4 K+ binding sites. Here, we report 2D IR spectra of a semisynthetic KcsA channel with site-specific 13C18O isotope labels in the selectivity filter. The ultrafast time-resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by MD simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.

Potassium channels are K+-selective protein pores in cell membrane. The selectivity filter is the functional unit that allows K+ channels to distinguish potassium (K+) and sodium (Na+) ions. The filter's structure depends on whether K+ or Na+ ions are bound inside it. We synthesized a K+ channel containing the d-enantiomer of alanine in place of a conserved glycine and found by x-ray crystallography that its filter maintains the K+ (conductive) structure in the presence of Na+ and very low concentrations of K+. This channel conducts Na+ in the absence of K+ but not in the presence of K+. These findings demonstrate that the ability of the channel to adapt its structure differently to K+ and Na+ is a fundamental aspect of ion selectivity, as is the ability of multiple K+ ions to compete effectively with Na+ for the conductive filter.