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.
Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA.
Alexander G
AG
Komarov
Kellie M
KM
Linn
Jordan J
JJ
Devereaux
Francis I
FI
Valiyaveetil
D000595:Amino Acid Sequence; D004594:Electrophysiology; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015221:Potassium Channels / Q000138:chemical synthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010455:Peptides; D015221:Potassium Channels; D011994:Recombinant Proteins