title: In vitro folding of KvAP, a voltage-gated K+ channel.
abstract: In this contribution, we report in vitro folding of the archaebacterial voltage-gated K(+) channel, K(v)AP. We show that in vitro folding of the K(v)AP 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(v)AP 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(v)AP channel provides a good model system for investigating the folding of a multidomain integral membrane protein.
authors: The Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon 97239, United States.
authors: Prasanna K
authors: PK
authors: Devaraneni
authors: Jordan J
authors: JJ
authors: Devereaux
authors: Francis I
authors: FI
authors: Valiyaveetil
mesh_terms: D041522:Aeropyrum / Q000737:chemistry*; D019843:Archaeal Proteins / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D018716:Molecular Mimicry; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding
chemical_list: D019843:Archaeal Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D024642:Potassium Channels, Voltage-Gated