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

Alexander G. Komarov, Kellie M. Linn, Jordan J. Devereaux, Francis Valiyaveetil

Research output: Contribution to journalArticle

29 Citations (Scopus)

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, β-(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.

Original languageEnglish (US)
Pages (from-to)1029-1038
Number of pages10
JournalACS Chemical Biology
Volume4
Issue number12
DOIs
StatePublished - Dec 18 2009

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Tryptophan
Membrane Proteins
Hydrogen
Peptides
Solid-Phase Synthesis Techniques
Proteolysis
Cysteine
Proteins
Purification
Hydrogen bonds
Membranes
Functional analysis
Electronic properties
Alanine
Structural properties
Fusion reactions
3-benzothienylalanine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

Modular strategy for the semisynthesis of a K+ channel : Investigating interactions of the pore helix. / Komarov, Alexander G.; Linn, Kellie M.; Devereaux, Jordan J.; Valiyaveetil, Francis.

In: ACS Chemical Biology, Vol. 4, No. 12, 18.12.2009, p. 1029-1038.

Research output: Contribution to journalArticle

Komarov, Alexander G. ; Linn, Kellie M. ; Devereaux, Jordan J. ; Valiyaveetil, Francis. / Modular strategy for the semisynthesis of a K+ channel : Investigating interactions of the pore helix. In: ACS Chemical Biology. 2009 ; Vol. 4, No. 12. pp. 1029-1038.
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