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KCNE3 is a potassium channel accessory transmembrane protein that regulates the function of various voltage-gated potassium channels such as KCNQ1. KCNE3 plays an important role in the recycling of potassium ion by binding with KCNQ1. KCNE3 can be found in the small intestine, colon, and in the human heart. Despite its biological significance, there is little information on the structural dynamics of KCNE3 in native-like membrane environments. Molecular dynamics (MD) simulations are a widely used as a tool to study the conformational dynamics and interactions of proteins with lipid membranes. In this study, we have utilized all-atom molecular dynamics simulations to characterize the molecular motions and the interactions of KCNE3 in a bilayer composed of: a mixture of POPC and POPG lipids (3:1), POPC alone, and DMPC alone. Our MD simulation results suggested that the transmembrane domain (TMD) of KCNE3 is less flexible and more stable when compared to the N- and C-termini of KCNE3 in all three membrane environments. The conformational flexibility of N- and C-termini varies across these three lipid environments. The MD simulation results further suggested that the TMD of KCNE3 spans the membrane width, having residue A69 close to the center of the lipid bilayers and residues S57 and S82 close to the lipid bilayer membrane surfaces. These results are consistent with previous biophysical studies of KCNE3. The outcomes of these MD simulations will help design biophysical experiments and complement the experimental data obtained on KCNE3 to obtain a more detailed understanding of its structural dynamics in the native membrane environment.

One of the major challenges in solubilization of membrane proteins is to find the optimal physiological environment for their biophysical studies. EPR spectroscopy is a powerful biophysical technique for studying the structural and dynamic properties of macromolecules. However, the challenges in the membrane protein sample preparation and flexible motion of the spin label limit the utilization of EPR spectroscopy to a majority of membrane protein systems in a physiological membrane-bound state. Recently, lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have emerged as a membrane mimetic system for investigating the structural studies of membrane proteins. However, its detail characterization for membrane protein studies is still poorly understood. Recently, we characterized the potassium channel membrane protein KCNQ1 voltage sensing domain (KCNQ1-VSD) and KCNE1 reconstituted into lipodisq nanoparticles using EPR spectroscopy. In this study, the potassium channel accessory protein KCNE3 containing flexible N- and C-termini was encapsulated into proteoliposomes and lipodisq nanoparticles and characterized for studying its structural and dynamic properties using nitroxide based site-directed spin labeling EPR spectroscopy. CW-EPR lineshape analysis data indicated an increase in spectral line broadenings with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated an enhanced quality of distance measurements with an increase in the phase memory time (T m) values upon incorporation of the sample into lipodisq nanoparticles, when compared to proteoliposomes. These results agree with the solution NMR structural structure of the KCNE3 and EPR studies of other membrane proteins in lipodisq nanoparticles. This study along with our earlier studies will provide the reference characterization data that will provide benefit to the membrane protein researchers for studying structural dynamics of challenging membrane proteins. [Display omitted] • Lipodisq nanoparticles have emerged as a membrane mimetic system for investigating the structural studies of membrane proteins. • Structural dynamics of KCNE3 was characterized in the presence of Lipodisq nanoparticles using EPR spectroscopy. • DEER measurements showed improvements in the quality of distance measurements with an increase in the phase memory time (T m) in the presence of lipodisq nanoparticles compared to liposomes. [ABSTRACT FROM AUTHOR]

KCNE3 is a potassium channel accessory transmembrane protein that regulates the function of various voltage-gated potassium channels such as KCNQ1. KCNE3 plays an important role in the recycling of potassium ion by binding with KCNQ1. KCNE3 can be found in the small intestine, colon, and in the human heart. Despite its biological significance, there is little information on the structural dynamics of KCNE3 in native-like membrane environments. Molecular dynamics (MD) simulations are a widely used as a tool to study the conformational dynamics and interactions of proteins with lipid membranes. In this study, we have utilized all-atom molecular dynamics simulations to characterize the molecular motions and the interactions of KCNE3 in a bilayer composed of: a mixture of POPC and POPG lipids (3:1), POPC alone, and DMPC alone. Our MD simulation results suggested that the transmembrane domain (TMD) of KCNE3 is less flexible and more stable when compared to the N- and C-termini of KCNE3 in all three membrane environments. The conformational flexibility of N- and C-termini varies across these three lipid environments. The MD simulation results further suggested that the TMD of KCNE3 spans the membrane width, having residue A69 close to the center of the lipid bilayers and residues S57 and S82 close to the lipid bilayer membrane surfaces. These results are consistent with previous biophysical studies of KCNE3. The outcomes of these MD simulations will help design biophysical experiments and complement the experimental data obtained on KCNE3 to obtain a more detailed understanding of its structural dynamics in the native membrane environment.