TY - JOUR
T1 - In vitro folding of K vAP, a voltage-gated K + channel
AU - Devaraneni, Prasanna K.
AU - Devereaux, Jordan J.
AU - Valiyaveetil, Francis I.
PY - 2011/12/6
Y1 - 2011/12/6
N2 - 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.
AB - 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.
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U2 - 10.1021/bi2012965
DO - 10.1021/bi2012965
M3 - Article
C2 - 22044112
AN - SCOPUS:82455175406
SN - 0006-2960
VL - 50
SP - 10442
EP - 10450
JO - Biochemistry
JF - Biochemistry
IS - 48
ER -