Semisynthesis and folding of the potassium channel KcsA.
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.
Valiyaveetil
Francis I
FI
Howard Hughes Medical Institute, The Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, New York, New York 10021, USA.
MacKinnon
Roderick
R
Muir
Tom W
TW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002096:C-Peptide / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry; D012604:Scorpion Venoms / Q000737:chemistry; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D002096:C-Peptide; D008565:Membrane Proteins; D015221:Potassium Channels; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C107790:prokaryotic potassium channel; C087603:agitoxin 2