Semisynthesis and folding of the potassium channel KcsA

Francis Valiyaveetil, Roderick MacKinnon, Tom W. Muir

Research output: Contribution to journalArticle

126 Citations (Scopus)

Abstract

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 α-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 α-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 α-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.

Original languageEnglish (US)
Pages (from-to)9113-9120
Number of pages8
JournalJournal of the American Chemical Society
Volume124
Issue number31
DOIs
StatePublished - Aug 7 2002
Externally publishedYes

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Potassium Channels
Potassium
Ligation
Proteins
Peptides
Inteins
Glutathione Transferase
Membrane Proteins
Fusion reactions
Membranes
Amino acids
Amino Acids
C-Peptide
Inclusion Bodies
Polypeptides
Lipids

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Semisynthesis and folding of the potassium channel KcsA. / Valiyaveetil, Francis; MacKinnon, Roderick; Muir, Tom W.

In: Journal of the American Chemical Society, Vol. 124, No. 31, 07.08.2002, p. 9113-9120.

Research output: Contribution to journalArticle

Valiyaveetil, Francis ; MacKinnon, Roderick ; Muir, Tom W. / Semisynthesis and folding of the potassium channel KcsA. In: Journal of the American Chemical Society. 2002 ; Vol. 124, No. 31. pp. 9113-9120.
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