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

Abstract

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.

Fig. 1. The modular semi-synthesis of the KcsA channel

The KcsA polypeptide is synthesized by two sequential ligation reactions. The first ligation reaction between a recombinantly expressed C-peptide and a chemically synthesized region of interest (ROI) peptide yields the intermediate peptide. The Thz protecting group (green sphere) on the N-terminal Cys of the intermediate peptide is removed and the de-protected intermediate peptide is ligated to a recombinantly expressed N-peptide thioester to yield the KcsA polypeptide. The KcsA polypeptide is folded in vitro to the native state. The modular strategy can be used to assemble semi-synthetic KcsA channels in which the selectivity filter (left) or the pore helix (right) is obtained by chemical synthesis. The protein segments obtained by chemical synthesis are colored red while the protein segments obtained by recombinant means are colored grey. The ligation sites are represented by yellow spheres.

Fig. 2. The Sumo fusion-proteolysis strategy for obtaining membrane spanning N-Cys peptides

a) The peptide of interest is expressed as a sumo fusion. The fusion protein is cleaved by the sumo protease to generate the desired N-Cys peptide that is purified by RP-HPLC. b) SDS-PAGE of the sumo fusion protein (lane 1) and the proteolysis reaction (lane 2) with P, sumo protease; S, sumo protein; C, N-Cys KcsA (82–160) C-peptide. c) ES-MS of the purified N-Cys KcsA peptide (82–160). Inset, reconstructed spectrum (Expected mass = 10220.6).

Fig. 3. Semi-synthesis of KcsA^(70,82)

a) SDS-PAGE of the first ligation reaction between the C-peptide (C, residues 82–160) and the filter peptide (residues 70–81) to form the intermediate peptide (I, residues 70–160) at 0 min (lane 1) and 24 h (lane 2). b) ES-MS of the intermediate peptide. Inset, reconstructed spectrum (Expected mass = 11431.9). c) SDS-PAGE of the second ligation reaction between the N-peptide thioester (N, residues 1–69) and the intermediate peptide to form the KcsA polypeptide at 0 min (lane 1) and 24 h (lane 2). d) SDS-PAGE showing the folding of semi-synthetic KcsA by lipids. The unfolded monomeric (M, which corresponds to the KcsA polypeptide, P) and the folded tetrameric KcsA (T) are indicated.

Fig. 4. Single channel activity of semi-synthetic KcsA

Single channel traces for the wild-type (WT) KcsA channel, the semi-synthetic KcsA^(70,82) and KcsA^(54,70) channels recorded at +150 mV in 10 mM succinate/150 mM KCl (pH 4.0) inside and 10 mM HEPES/150 mM KCl (pH 7.5) outside are shown.

Fig. 5. The KcsA(Trp68→3BT) channel

a) Close-up view of the selectivity filter. The peptide backbone of residues 68–80 and the side chains of residues Trp68 and Thr72 from one subunit and Tyr78 from an adjacent channel subunit that are involved in a H-bond network are shown in stick representation (pdb code: 1k4c). K⁺ ions are shown as magenta spheres. b) Representative single channel traces of the KcsA(Trp68→3BT) channels recorded at +150 mV in 10 mM succinate/150 mM KCl (pH 4.0) inside and 10 mM HEPES/150 mM KCl (pH 7.5) outside. c) Single channel current at +150 mV as a function of the K⁺ ion concentration for KcsA(Trp68→3BT) (Δ) and wild-type KcsA(□). Solid lines have no theoretical meaning. d) Macroscopic currents recorded using 10 mM succinate and 150 mM KCl (pH 4.0) as the internal solution, and 10 mM HEPES, 20 mM KCl, and 130 mM NaCl (pH 7.5) as the external solution are plotted against the voltage applied to determine the reversal potential (calculated reversal potential = −51 mV, measured = −48.3±2.2 mV).

Fig. 6. Rb⁺ conduction by KcsA(Trp68→3BT) channels

a) Representative single channel traces of the KcsA(Trp68→3BT) or wild-type KcsA channels recorded at +150 mV in 10 mM succinate/150 mM RbCl (pH 4.0) inside and 10 mM HEPES/150 mM RbCl (pH 7.5) outside. b) Single channel current at +150 mV as a function of the Rb⁺ ion concentration for KcsA(Trp68→3BT) (Δ) and wild-type KcsA(□). Solid lines have no theoretical meaning.