In vitro folding of K vAP, a voltage-gated K + channel

Prasanna K. Devaraneni, Jordan J. Devereaux, Francis Valiyaveetil

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

In this contribution, we report in vitro folding of the archaebacterial voltage-gated K + channel, K vAP. We show that in vitro folding of the K vAP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature, and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature, and almost quantitative refolding of the K vAP channel is observed at 80 °C. To differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition, suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the K vAP channel provides a good model system for investigating the folding of a multidomain integral membrane protein.

Original languageEnglish (US)
Pages (from-to)10442-10450
Number of pages9
JournalBiochemistry
Volume50
Issue number48
DOIs
StatePublished - Dec 6 2011

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Voltage-Gated Potassium Channels
Temperature
Assays
Chemical analysis
Lipids
Protein Unfolding
Lipid bilayers
Lipid Bilayers
Cysteine
Membrane Proteins
In Vitro Techniques
Proteins

ASJC Scopus subject areas

  • Biochemistry

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In vitro folding of K vAP, a voltage-gated K + channel. / Devaraneni, Prasanna K.; Devereaux, Jordan J.; Valiyaveetil, Francis.

In: Biochemistry, Vol. 50, No. 48, 06.12.2011, p. 10442-10450.

Research output: Contribution to journalArticle

Devaraneni, Prasanna K. ; Devereaux, Jordan J. ; Valiyaveetil, Francis. / In vitro folding of K vAP, a voltage-gated K + channel. In: Biochemistry. 2011 ; Vol. 50, No. 48. pp. 10442-10450.
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