Investigation of Structural Dynamics of the KCNE3 in a Lipid Bilayer Membrane using Site-Directed Spin Labeling EPR Spectroscopy

Abstract

KCNE3, a single pass membrane protein, is responsible for controlling the function of multiple potassium channels, one of which is the KCNQ1 channel. The binding of KCNE3 with KCNQ1 is vital in transepithelial potassium ion recycling which has been associated with disorders such as cystic fibrosis. Although KCNE3 has great biological significance, its structural and dynamic information in a native like environment is poorly understood. In this study, we have utilized site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) spectroscopic technique to investigate structural dynamics of KCNE3 in lipid bilayers. KCNE3 was overexpressed and purified in LMPG detergent micelles. MTSL spin label was introduced followed by the incorporation of the protein into POPC/POPG lipid bilayer. Continuous wave (CW)-EPR spectral lineshape analysis was performed on several spin labeling sites including transmembrane domain and extracellular domain of KCNE3 in LMPG detergent micelles and POPC/POPG lipid bilayes to obtain the spin label sidechain mobility and the rotational correlation times. Our EPR data indicated that the nitroxide spin label sidechains in liposomes have slower motion for the sites in the transmembrane domain of KCNE3 when compared to that in the extracellular region of KCNE3. Our results are consistent with the previously published solution NMR structure of KCNE3.